Advances in Microbiology, 2015, 5, 787-796
Published Online November 2015 in SciRes. http://www.scirp.org/journal/aim
http://dx.doi.org/10.4236/aim.2015.512083
How to cite this paper: Batta, Y.A. (2015) Production and Testing of Biopesticide for Control of Postharvest Mold Infections
on Fresh Fruits of Apple and Pear. Advances in Microbiology, 5, 787-796. http://dx.doi.org/10.4236/aim.2015.512083
Production and Testing of Biopesticide for
Control of Postharvest Mold Infections on
Fresh Fruits of Apple and Pear
Yacoub A. Batta
Laboratory of Plant Protection, Department of Plant Production & Protection, Faculty of Agriculture &
Veterinary Medicine, An-Najah National University, Nablus, West Bank, Palestine
Received 8 October 2015; accepted 13 No vembe r 2015; published 16 November 2015
Copyright © 2015 by author and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/
Abstract
The present research aimed at producing a biopesticide with proper formulation of invert emul-
sion (water-in-oil type) and effective strain of Trichoderma harzianum then, testing it against pa-
thogens of postharvest mold infections (Botrytis cinerea & Penicillium expansum) on fresh fruits of
apple and pear. The proper formulation of invert emulsion (IE#3) used for biopesticide produc-
tion has the following ingredients (100% w/w): soybean oil (28.50%), coconut oil (19.50%), oil-
soluble emulsifier: Tween 20 (2.0%), glycerine (4.25%), water-soluble emulsifier: dehymuls k
(0.75%), sterile distilled water (22.5%) and conidial suspension of the effective strain of T. har-
zianum “TrichoPAL1” in water (22.5%, concentration 1 × 107 conidia/ml). Testing efficacy of the
produced biopesticide has indicated a significant reduction in the disease lesion diameter of mold
infections on wounded apple and pear fruits stored at 20˚C ± 1˚C compared to the untreated fruits
or control (reduction from up to 38.75 to about 7.50 mm, respectively, according to the type of
mold infections and fruit type). Also, the treatment with the produced biopesticide has resulted in
a long protection period from mold infections on wounded and un-wounded fresh fruits of apple
and pear stored under controlled and semi-commercial conditions (up to 2.5 months according to
the type of mold infections and fruit type). In conclusion, the overall results have demonstrated
the effectiveness of produced biopesticide on stored fruits under controlled and semi-commercial
conditions therefore, it is recommended to test this effectiveness on marketed fruits stored under
variable conditions before applying it at a large scale.
Keywords
Apple Fruit, Pear Fruit, Trichoderma harzianum, Botrytis cinerea, Penicillium expansum,
Biop esticides, Postharvest Mold Infections

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788
1. Introduction
The control of postharvest mold infections on fresh fruits is usually done by the conventional use of synthetic
fungicides [1] [2]. Alternative approaches to chemical use are highly appreciated due to the increasing importance
of integrated management strategies relying on biocontrol possibly combined with selective application of fun-
gicides [3] [4]. The above strategies must achieve two demands: effectiveness and environmental safety. Also,
integrated control could be a convenient strategy for newer attempts in the control of postharvest mold infections
[5] [6].
Regarding the group of interesting biocontrol agents that can be involved in the integrated management
strategies, the antagonistic fungi are considered the most promising agents because they are currently being de-
veloped as a novel approach for the control of many plant diseases [7]. However, these fungi suffer from the
following disadvantag es: i) their initial or acute biocontrol potential is much slower in comparison with chemical
fungicides; ii) they need hi gh hum idity for coni dial ge rmi nation an d subse quent de velo pment and spo rulat ion; iii)
they are susceptible to UV irradiation; and iv) some targeted plant diseases may develop defence mechanisms
against the fungal attack. Some of these constraints could be overcome by developing, for example, formulations
which improve the stability of these fungi, UV resistance, distribution on the surface of disease host-plant, and
contact to the targets or can synergistically enhance efficacy by improving the virulence of these fungi [8] [9].
The use of antago nistic fu ngi as bioc ontrol a gents of pl ant diseases, for example, postharvest mold infections,
has been previously reported by some investigators [10]-[13], but without attempting to formulate these bio-
control agents during their application. Therefore, formulation of these fungi is crucial for increasin g the ir ef-
ficacy during application against targeted plant diseases. Invert Emulsions (water-in-oil type) are one of the
promising formulations that can be used for antagon istic fungi because these emulsions contain the necessary
water f or germinat ion of fungal conidia and their development during or after application. The water introduced
into emulsions is usually homogenized with oil at a high speed (20,000 rpm for 1.5 minutes using homogeniser),
thus the water droplets in the final emulsions are completely surrounded by the oil droplets (water-in-oil
emulsion). Therefore, oil droplets prevent the water droplets from quick evaporation. Under hot dry conditions,
water used for preparation of the conidial suspension of these fungi will evaporate and dry up quickly but if this
water is incorporated into invert emulsions as described above, it will evaporate very slowly following appli-
cation due to the above reason [14].
Recently, research studies conducted in our laboratory under controlled environmental conditions have shown
that certain strains of Trichoderma harzianum applied i n in vert em ulsi on are prove d to be e ffect ive agai nst f ungal
plant pathogens infecting the fresh produce of various crops including fruits postharvest [15]-[17]. Therefore, in
the present research, biopesticides based on using new combinations (formulations) of invert emulsions with ef-
fective strain(s) of th e antagonistic fungu s—T. harzianum will be produced then tested against postharvest mold
infections on apple and pear fruits stored under variable environmental conditions and in semi-commercial con-
ditions. Therefore, the main objective of the present research was to produce an effective biopesticide against
postharvest mold infections on apples and pears and then test its effectiveness. To arrive this objective, the fol-
lowing aims should be fulfilled: i) screening for selec tion of the most effective str ain of Trich ode rma harzia num
against postharvest mold infections; ii) formulation of the selected strain of T. harzianum using ne w com binations
of invert emulsion formulatio n; iii) testing the efficacy of the formulated strain of T. ha rzianum in invert emul-
sions against mold infections especially those ca use d by Botrytis cinerea and Penicillium expansum on apple and
pear fruits stored in incubator or under semi-commercial conditions.
2. Materials and Methods
2.1. Isolation of Pathogens Causing Mold Infections on Fresh Fruits of Apple and Pear
The following postharvest mold infections of fruits were studied in the present research: Gray mold caused by
Botrytis cinerea and Blue mold caused by Penicillium expansum. Local strains of B. cinerea (designated
BCPAL) and P. expansum (designated PEPAL) were isolated on Potato Dextrose Agar medium (PDA) amended
with Chloramphenicol antibiotic (250 mg/L W/V, added to the medium after autoclaving). The two strains were
isolated from naturally infected fruits of apple (source: a local wholesale market). Pure cultures of these isolated
strains were used after sub-culturing of them on the same culture medium (PDA). These cultures were kept in a
cold room at 4˚C ± 1˚C for being used during bioassays’ conduction.

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2.2. Isolation of Strains of the Antagonistic Fungus: T. harzianum
Local strain s of T. harzianum were used in the present bioassays since this fungus has an antagonistic effect to
many causal agents of fungal plant diseases including postharvest mold fungi of fresh fruits. Four local strains of
T. harzianum were isolated from dead tomato roots previously infected with root rot fungi and then buried in the
soil cultivated with various crops in Tulkarm district, Palestine. The isolated strains were designated “TrichoP-
AL1”, “TrichoPAL2”, “TrichoPAL3” and “TrichoPAL4”. They were first isolated on Potato D extrose Agar me-
dium (PDA) amended with Chloramphenicol antibiotic (250 mg/L W/V, added to the medium after autoclaving).
Identification of these strains as isolates of T. harzianum was made according to the typical morphological cha-
racteristics of this fungus described by Grondona et al. 1997. After sub-culturing of these strains on oatmeal
agar medium (OMA), pure cultures of these strains were kept in a cold room (4˚C ± 1˚C) for being used during
bioassays’ conduction.
2.3. Screening Test for the Efficacy of Isolated T. harzianum Strains
Efficacy of the isolated strains of T. harzianum was tested against postharvest mold infections for screening the
most effective strain. For this purpose, healthy apple and pear fruits were infected with B. cinerea (strain
BCPAL) and P. expans um (strain PEPAL) then treated with the conidial suspension of T. harzianum is olates at a
concentration of 1 × 107 conidia/ml each. Comparison of the efficacy of these strains was done according to the
suppression level of the disease lesion diameter resulted from B. cinerea and P. expansum infection on fruits
used in bioassays. The infection with B. cinerea and P. expansum on apple and pear fruits and then treating them
with the antagonistic fungus (T. harzianum) was done by depositing 25 µl-droplet of conidial suspension of B.
cinerea or P. expansum (concentration 1 × 106 conidia /ml) per fruit after inducing a small superficial wo und on
each fruit surface. A micropipette (100 µl capacity) is used for depositing of each droplet on each fruit. The con-
trol treatment is consisting of superficially wounded fruits treated with 25 µl-droplet of sterile distilled water.
Inoculated and control fruits were then treated with conidial suspension of the following T. harzianum strains:
“TrichoPAL1”, “TrichoPAL2”, “Tri choPAL3” and “TrichoPAL4”. This treatment was done by depositing 25
µl-droplet of conidial suspension (concentration 1 × 107 conidia/ml each) of each strain per fruit at the same site
of disease inoculation using a 100 µl-micropipette. Four fruits represen ting 4 replicates per inoculated fruit type
per pathogen per T. harzianum strain or control treatment were used. Each fruit was kept in a closed plastic pot
of 9 cm diameter and 15 cm depth. A completely randomized design was used. The disease lesion diameter ob-
tained 7 days after the treatment with the antagonistic fungus and incubation at 20˚C ± 1˚C was measured on
each fruit then the mean lesion diameter of each disease on each fruit type per disease per T. harzianum strain
was calculated. Finally, the Average mean of disease lesion diameter per strain of T. harzianum was calculated
and used for comparison of the efficacy of tested strains to select the most effective one.
2.4. Preparation of Invert Emulsion Formulations and Selection of the Most Proper
New combinations of invert emulsion formulations (water-in-oil type) were prepared in this research. Each com-
bination was mainly consisted of two phases: i) aqueous or water phase comprising of sterile distilled water,
glycerine and water-soluble emulsifier, and ii) oil phase comprising of plant-origin oil(s) and oil-soluble emulsi-
fier. The propo rtion of each p hase in t he fina l invert emulsion was 50% (w/w) a nd the percentage of ingredients in
each phase was changed during preparation so that the resulting combination was accepted as a suitable formu-
lation if it had a high stability and low viscosity. Mixing of t he tw o phase s du ring pr epara tion o f the e muls ion was
done at a high spe ed (20,00 0 rpm for 1.5 minut es using a hom ogenizer). T his is done to en sure the homogenei ty of
the emulsion. Five combinations were prepared using the above technique in order to select the most proper one
characterized by the highest stability and lowest viscosity. The stability of the prepared emulsion was judged by
the non-separa ti on of t he t wo pha ses com posi ng the em ul sio n alo ng the tim e foll owi ng pre para tio n. The vi scosi ty
of the pre pare d emulsi on wa s m ea sure d by a vi scometer. Th e em ul si on is c ons i dere d “n ot vi sco us” i f i ts vi sc osit y
<25 cps (centipoises), “relatively viscous” if its viscosity 25 - 35 cps and viscous if its viscosity >35 cps.
2.5. Introduction of the Most Effective Strain of T. harzianum into the Selected
Formulation of Invert Emulsion
The most effective strain of T. harzianum obtained by screening in the above section was introduced into the se-

Y. A. Batta
790
lected formulation of invert emulsion characterized by the highest stability and lowest viscosity. Introduction was
done, at first, into the aqueous or water phase which finally become consisted of sterile distilled w ater, conidial
suspension of the effective strain of T. harzianum, glycerine an d w ater-solub le emulsifier. Th e ingredients of oil
phase did not change and remained as indicated above. The ratio of these two phases in the final invert emulsion
was the same as indicated above. Conidial suspension of the most effective strain of T. harzianum in the f or mu-
lation was 1 × 107 conidia/ml. Mixing of the two phases after introduction of the conidial suspension of the an-
tagonistic fungus was done as indicated in the previous section to ensure the homogeneity of the emulsion.
2.6. Testing Efficacy of Formulated Strain of T. harzianum against Postharvest
Mold Infections on Fruits
The efficacy test was practiced against mold infections on wounded and unwounded fruits of apple and pear
stored at 20˚C ± 1˚C in the incubator or at 20˚C ± 5˚C under semi-commercial conditions as follows.
2.6.1. Efficacy on Wounded Fruits of Apple and Pear Stored in Incubator at 20˚C ± 1˚C
To test efficacy of the most effective str ain of T. harzianum formulated in invert emulsion against pathogens of
postharvest mold infections, healthy fruits of apple and pear were, first, infected with B. cinerea and P. expan-
sum then, treated with the formulated T. harzianum according to the protocol indicated in the previous section.
Comparison of the efficacy after the treatment was done according to the suppression level of the disease lesion
diameter resulted from B. cinerea or P. expansum infection on treated fruits. The technique of inoculation with
the mold pathogens on apple and pear fruits and then treatment with the formulated T. harzianum was done by
depositing 25 µl-droplet of conidial suspension of each one of mold pathogens (concentration 1 × 106 conidia/ml)
per fruit after inducing a small circular superficial wou nd (2 mm diameter) on each fruit surf ace. A micropipette
(100 µl capacity) was used for depositing the droplet on the wounded fruit. The control treatment is consisting
of superficially wounded fruits treated with 25 µl-droplet of sterile d istilled water. Inoculated and control fruits
were then treated with 25 µl-droplet of formulated strain of T. harzianum (concentration 1 × 107 conidia/ml each)
deposited at the same site of disease inoculation. In each bioassay, four types of treatments were used per pa-
thogen per fruit type. The treatments were: formulated T. harzianum in invert emulsion, unformulated T. har-
zianum, blank formulation of invert emulsion and control (untreated with T. harzianum). Four fruits representing
4 replicates per treatment type were u sed. After that, each treated fruit was placed in a closed p lastic pot (9 cm
diameter and 15 cm depth) then stored in an incubator at 20˚C ± 1˚C. A completely randomized design was used.
For each pathogen on each fruit type, the disease lesion diameter obtained 7 days after the treatment was meas-
ured then the mean lesion diameter of each treatment was calculated then used for comparison of the efficacy.
2.6.2. Efficacy on Un-Wounded Fruits of Apple and Pear Stored in Incubator at 20˚C ± 1˚C
To test efficacy of the most effective strain of T. harzianum formulated in invert emulsion against the fungal
pathogens of postharvest mold infections, healthy un-wounded fruits of apple and pear were, first, infected with
B. cinerea and P. expansum then treated with the formulate d T. harz ianum strain. Evaluation of the efficacy was
done according to the time required for the appearance of disease symptoms (B. cinerea or P. expansum) on un-
treated and treated fruits with the formulated T. harzianum. The technique of inoculation with the fungal patho-
gens of mold infections on apple and pear fruits was done b y spraying 200 µl spray volume of conidial suspen-
sion of these fungal pathogens (concentration 1 × 106 conidia/ml) per healthy un-wounded fruit then the inocu-
lated fruits were placed in rectangular plastic boxes (20 × 10 × 10 cm; long, wide and high, respectively). A
transparent plastic film was used to cover each box. A small calibrated hand sprayer (1 L capacity) was used for
spraying the intended spray volume on the un-wounded fruits. The control treatment is consisting of un-
wounded fruits sprayed with the same quantity of spray volume of sterile distilled water after infection with
mold pathogens. Inoculated and control fruits were then sprayed with 200 µl spray volume of formulated T. har-
zianum (concentration 1 × 107 conidia/ml each) per fruit in the same manner as indicated above. In each bioas-
say, four types of treatments were used per pathogen per fruit type. The treatments were: formulated T. harzia-
num in invert emulsion, unformulated T. harzianum, blank formulation of invert emulsion and control (untreated
with T. harzianum). Five fruits per box representing 5 replicates per treatment type were used. A completely
randomized design was used. The time needed for the appearance of typical symptoms of each disease (gray or
blue mold) on fruits of each treatment was determined and then used for evaluation of the efficacy. The plastic

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boxes containing fruits used for bioassays were stored in an incubator at 20˚C ± 1˚C until the appearance of the
disease lesion on the incubated fruit s.
2.6.3. Efficacy on Un-Wounded Fruits of Apple and Pear Stored at 20˚C ± 5˚C
(Under Semi-Commercial Conditions)
To test efficacy of the effective strain of T. harzianum formulated in invert emulsion against the fungal patho-
gens of postharvest mold infections on apple and pear fruits stored under semi-commercial conditions (20˚C ±
5˚C), rectangular plastic boxes (20 × 10 × 10 cm; long, wide and high, respectively) were used. A transparent
plastic film was used to cover the fruits after treatment. These boxes are similar to those used for marketing of
these fruits. The technique of inoculation with the fungal pathogens of mold infections on apple and pear fruits
was done by spraying 1.0 ml of conidial suspension of these fungal pathogens (concentration 1 × 106 conidia /
ml) on healthy un-wounded fruits kept in each plastic box. A small calibrated hand sprayer (1 L capacity) was
used for spraying the intended spray volume per box containing 5 healthy un-wounded fruits. The control treat-
ment is consisting of 5 healthy un-wounded fruits kept in one box and sprayed with the same spray volume of
sterile distilled water (1.0 ml). The inoculated and control fruits were then sprayed with 1.0 ml spray volume of
formulated T. harzianum (concentration 1 × 107 conidia/ml each) using the technique and equipment as indi-
cated above. For each bioassay, two types of treatments were used per pathogen per fruit type. The treatments
were: formulated T. har zianum in invert emulsion and control (untreated with T. harzianum ). Fiv e fruits pe r box
representing 5 replicates per treatment type were used. A completely randomized design was used. The time
needed for the appearance of typical symptoms of each disease (gray or blue mold) on fruits after the treatment
was determined and then used for evaluation of the efficacy. The protection period represents the time extending
from inoculation to appearance of the first symptoms of infection with the disease on treated fruits. During bio-
assays, the plastic bo xes that contain fruits were stored in a store-room at 20˚C ± 5˚C until the appearance of the
disease symptoms on incubated fruits. These conditions are similar to those used for storing fruits of apple and
pear during marketing of the product.
2.7. Statistical Analyses
For the targeted diseases of mold infections, the mean % of disease lesion diameter on fruits treated with the
formulated and unformulated T. harzianum was statistically analyzed using analysis of variance (ANOVA).
Significant differences (at P = 0.05) between the treatment means were determined by F-test. Also, the mean
separation was performed using Duncan’s Multiple Range test.
3. Results
3.1. Efficacy of the Isolated Strains of T. harzianum
As a result of the screening test, the strain of T. harzianum “TrichoPAL1 ” has shown the highest efficacy
against B . cinerea (strain BCPAL) and P. expansum (strain PEPAL) in comparison with other strains of T. har-
zianum because this strain has significantly redu ced (at P = 0.05) the average mean of disease lesion diameter of
both pathogens on wounded apple and pear fruits from 21.25 mm diameter (in the untreated control) to 12.31
mm diameter (Ta ble 1). Therefore, it has been selected as the most effective strain of T. harzianum for being in-
troduced into the selected formulation of invert emulsion. The formulated strain of T. harzianum was then used
for further bioassays as biopesticide. However, no significant differences were obtained between the average
means of disease lesion diameter of both pathogens on wounded apple and pear fruits for the other strains of T.
harzianum “TrichoPAL2”, “TrichoPAL3” and “Tricho P AL4” and the untreated control (Table 1), so they are
not selected for further bioassays.
3.2. Selection of Invert Emulsion Formulation as a Carrier of T. harzianum
Among the different combinations (formulations) of invert emulsion prepared, the combination “IE # 3” wa s se-
lected as the most proper because it has shown a high stability for long time with no viscosity (Tab le 2), so tha t
it has been selected as a carrier of T. harzianum “strain TrichoPAL1” then tested as biopesticide against mold
infections on fresh fruits. The selected formulation (IE # 3) has the followi ng in gredients: i) a mixture of two oils
of plant-origin (soybean oil : 28.50% w/w and coc onut oil: 19.50% w/w); ii) oil-soluble emulsifier (Tween 20: 2.0%
w/w); iii) sterile distilled water (22.5% w/w); iv) conidial suspension of the selected strain of T. harzianum in

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Table 1. Disease lesions of Botrytis cinerea (strain BCPAL) and Penicillium expansum (strain PEPAL) formed on wounded
apple and pear fruits (varieties: Red Delicious-Starking and Bartlett, respectively) 7 days after inoculation and treatment with
four strains of Trichoderma harzianum (incubation of treated fruits was done in an incubator at 20˚C ± 1˚C).
Strains of
T. harzianum
Mean disease lesion diameter of B. cinerea and P. expansum (in m m)
observed on wounded apple and pear fruits when treated with T. harzianum*
B. cinerea
on Apple fruit B . cinerea on Pear fruit P. expansum on Apple fruit P. expansum on pear fruit
Average mean of disease
lesion diameter (±SEM)
per strain of T. harzianum**
TrichoPAL1 14.25 10.50 11.00 13.50 12.31 (±0.79) A
TrichoPAL2 20.75 17.50 20.00 19.25 19.37 (±0.61) B
TrichoPAL3 19.50 21.25 21.00 19.00 20.19 (±0.48) B
TrichoPAL4 20.25 18.50 19.75 20.50 19.50 (±0.41) B
Untreated Control 20.00 21.25 21.00 22.75 21.25 (±0.49) B
*Four frui ts of each fruit t ype representing 4 replicates per pat hogen treatment per strain were u sed. Therefore, 4 replicates were p erformed for each
test (20 tests were performed for the 4 strains + control). SEM: standard error of the mean or standard deviation of the mean which is = Standard
Deviation of original distribution (SD)/Square root of Sample Size (n); **Mean s fo llowed by th e sa me letter are no t sign ifi cantly d iffer ent at P = 0.05
using ANOV A and Duncan’s Multiple Range test.
Table 2. Combinations of invert emulsion formulation (IE) based on variable percentage of ingredients composing oil phase
and water phase of the emulsion.
Combination
number of invert
emulsion (IE)
% of ingredients (w/w) of water phase and oil phase
compos ing the formul ation of invert emulsion (IE) Stability of
prepared
emulsion
Viscosity of
prepared
emulsion
Soybean
oil Coconut oil Tween 20
Sterile
distilled
water
Conidial
suspension
of T. harzianum glycerin Dehymuls k
IE # 1 29.00 18.00 3.00 21.00 24.00 4.75 0.25 Not stable Relatively
viscous
IE # 2 28.50 19.00 2.50 22.00 23.00 4.50 0.50 Not stable Relatively
viscous
IE # 3 28.50 19.50 2.00 22.50 22.50 4.25 0.75 Stable for
long time Not viscous
IE # 4 28.50 19.00 2.50 23.00 22.00 4.00 1.00 Not stable viscous
IE # 5 29.00 18.00 3.00 23.00 22.00 4.00 1.00 Not stable Viscous
water (TrichoPAL1: 22.5% w/w), v) glycerine (4.25% w/w), and water-soluble emulsifier (Dehymuls k: 0.75%
w/w) (Table 2 ). The total % of these ingredients (w/w) is 100 which is equally divided between the water phase
and the oil phase (50% w/w each).
3.3. Testing the Efficacy of Formulated T. harzianum in Invert Emulsion against
Mold Infections on Fresh Fruits
3.3.1. On Wounded Fruits of Apple and Pear Stored in an Incubator at 20˚C ± 1˚C
The mean of disease lesion diameter of B. cinerea and P. expansum on wounded apple fruits has significantly
decreased (at P = 0.05) when treated with the formulated strain “Tricho PAL1” of T. harz ianum in invert emul-
sion (combination IE # 3). This decrease was ranged from 35.75 and 18.75 mm to 6.25 and 3.75 mm for B. ci-
nerea and P. expansum, respectively, in comparison with the control (Table 3). The same trend was obtained in
the results on wounded pear fruits (significant reduction in the mean of disease lesion diameter of both patho-
gens due to treatment with the formulated T. harzianum) (Table 3). However, no significant differences are
present between means of the disease lesion diameter of both diseases on both types of fruits regarding the two
types of control treatments (untreated fruit control and Blank formulation of invert emulsion) (Table 3. This
may indicate that no harmful effects may be caused by the ingredients of the selected formulation.

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793
Table 3. Disease lesions of Botrytis cinerea (strain BCPAL) and Penicillium expansum (strain PEPAL) formed on wounded
apple and pear fruits (variety: Red Delicious-Starking and Bartlett, respectively) after inoculation and treatment with unfor-
mulated and formulated Trichoderma harzianum (strain TrichoPAL1) in invert emulsion (incubation of the treated and con-
trol fruits was done in incubator at 20˚C ± 1˚C).
Treatments with
T. harzianum
and control
Mean lesion diameter (in mm) (±SEM) of B. cinerea
appeared after 5 days of inoculation and treatment* Mean lesion diameter (in mm) (±SEM) of P. expansum appeared after 7 days of inoculation and treatment*
on wounded apple fruit on wounded pear fruit on wounded apple fruit on wounded pear fruit
Formulated
TrichoPAL1 of
T. harzianum 6.25 (±0.42) A** 7.50 (±0.53) A** 3.75 (±0.45) A** 6.2 (±0.49) A**
Un-formulated
TrichoPAL1 of
T. harzianum 14.50 (±0.78) B 17.00 (±0.67) B 7.50 (±0.81) B 12.50 (±0.77) B
Blank formulation of
invert emulsion 31.25 (±0.69) C 34.00 (±0.75) C 16.25 (±0.85) C 18.75 (±0.67) C
Control
(untreated fruits) 35.75 (±(0.82) C 38.75 (±0.93) C 18.75 (±0.91) C 21.25 (±0.99) C
*Four frui ts of each fruit t ype representing 4 replicates per pat hogen treatment per strain were u sed. Therefore, 4 replicates were p erformed for each
test (16 tests were per formed f or formul ated st rain, un formulated strain, b lank for mulatio n and contr ol). SEM: standar d error of the mean or st andard
deviation of the mean which is = Standard Deviation of original distribution (SD)/Square root of Sample Size (n); **Wit hin each co lumn, mean s fol-
lowed by the same letter are not significantly different at P = 0.05 using ANOVA and D uncan's Multiple Range test.
3.3.2. On Un-Wounded Fruits of Apple and Pear Stored in an Incubator at 20˚C ± 1˚C
The time required for the appearanc e of the first symptoms of infection with B. cinerea and P. expan sum on un-
wounded fresh apples stored in an incubator under controlled conditions (20˚C ± 1˚C) was the longest on apple
fruits treated with the formulated T. harzianum (72 and 58 days for the two diseases, respectively) and the
shortest on apple fruits of the control treatment (12 and 18 days for the two diseases, respectively) (Table 4).
Intermediate time periods were required for the appearance of the first symptoms of infection with the
above-mentioned diseases on apples treated with the unformulated T. harzianum or with the blank formulation
(Table 4). These time periods represent the protection period on apple fruits needed to prevent infection with
these diseases when treated with the formulated T. harzianum compared with the control. Lower values of pro-
tection periods against the same mold inf ections (B. cinerea and P. expansum) on un-w ounded fresh pear fru its
stored at 20˚C ± 1˚C were obtained when treated w ith the formulated T. harzianum compared to the control (65
and 50 days versus 11and 19 days for the two di se a s e s , respectively) (Table 4).
3.3.3. On Un-Wounded Fruits of Apple and Pear Stored in Semi-Commercial
Conditions at 20˚C ± 5˚C
The time required for the appearance of the first symptoms of infection with B. cinerea and P. expansum on
un-wounded fresh fruits of apple stored in semi-commercial conditions (20˚C ± 5˚C) was the longest on apple
fruits treated with the formulated T. harzianum (61 and 44 days for the two diseases, respectively) and the
shortest on apple fruits of the control treatment (10 and 15 days for the two diseases, respectively) (Table 5).
These time periods represent the protection period on apple fruits needed to prevent infection with the diseases
when treated with the formulated T. harzianum compared with the control. Lower values of protection periods
against the same mold infections (B. cinerea and P . expansum) on un-wounded fresh pear fruits stored at 20˚C ±
5˚C were obtained when treated with the formulated T. harzianum compared to the control (50 and 40 days ver-
sus 9 and 12 day for the two diseases, respectively) (Table 5).
4. Discussion
Results obtained in the present research demonstrate the ability of the produced biopesticide to reduce signifi-
cantly the mold infections on fresh fruits of apple and pear stored under controlled and partially controlled con-
ditions. This ability is attributed to the capacity of the formulated strain of T. harzianum (TrichoPAL1) to sup-
press the growth of pathogens causing mold infections on treated fruits (B. cinerea and P. expansum) hence
control these postharvest diseases. In addition to this ability, the formulation of invert emulsion (water-in-oil
type) used for production of this biopesticide has significantly increased its efficacy against the targeted post

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Table 4. Time period needed for the first appearance of disease lesion of Botrytis cinerea (strain BCPAL) and Penicillium
expansum (strain PEPAL) on un-wounded apple and pear fruits (variety: Red Delicious-Starking and Bartlett, respectively)
treated with unformulated and formulated Trichoderma harzianum (strain TrichoPAL1) (incubation of the treated and con-
trol fruits was done in incubator at 20˚C ± 1˚C).
Treatments with
T. harzianum a nd c o nt r o l
Time needed for the first appearance
of lesions of B. cinerea (in days)* Time needed for the first appearance of lesions of P. expansum (in days)*
on un-wounded
apple fruit on un-wounded pear fruit on un-wounded apple fruit on un-wounded pear fruit
Formulated TrichoPAL1
of T. harzianum 72 65 58 50
Un-formulated TrichoPAL1
of T. harzianum 39 40 32 38
Blank formulation
of invert emulsion 14 12 17 16
Control (untreated fruits) 12 11 18 19
*Four frui ts of each fruit t ype representing 4 replicates per pat hogen treatment per strain were u sed. Theref ore, 4 replicates were performed for each
test (16 tests were performe d for formulated strain, unformulated strain, blank form ul ation and control).
Table 5. Time period needed for the appearance of the first disease lesion of Botrytis cinerea (strain BCPAL) and Penicil-
lium expansum (strain PEPAL) on un-wounded apple and pear fruits (variety: Red Delicious-Starking and Bartlett, respec-
tively) treated with formulated Trichoderma harzianum (strain TrichoPAL1) (incubation of the treated and control fruits was
done in semi-comm er cial co nditions (20˚C ± 5˚C)).
Treatments with
T. harzianum a nd c o nt r o l
Time needed for the first appearance
of lesions of B. cinerea (in days)* Time needed for the first appearance of lesions of P. expansum (in days)*
on un-wounded
apple fruit on un-wounded pear fruit on un-wounded apple fruit on un-wounded pear fruit
Formulated TrichoPAL1
of T. harzianum 61 50 44 40
Control (untreated fruits) 10 9 15 12
*Four frui ts of each fruit t ype representing 4 replicates per pat hogen treatment per strain were u sed. Therefore, 4 replicates were p erformed for each
test (8 tests were performed for formulated strain and contr ol).
harvest mold infections when compared with the unformulated form of the strain. The reason for increasing this
efficacy is explained by selection of certain ingredients for b eing introduced into the formulation. For example,
a mixture of two oils of plant-origin: soybean oil (28.50% w/w) and coconut oil (19.50% w/w) characterized by
having a hi gh content of m onounsat urated fat suitable fo r preparing a stable inve rt emulsi on is used. In addition to
using two types of emulsifiers: oil-soluble emulsifier (T ween 20: 2.0% w/w) and water-soluble emulsifier (De-
hymul s k: 0.75% w/w) for producing a homogenous and stable invert emulsion. Glycerine (4 .2 5% w/w) was also
used for decreasing the activity of wa ter molecule in th e emulsion by preventing its active evaporation. A similar
enhancement of the efficacy has been reported for other strains of T. harzianum when introduced into invert
emulsion. For example, T. harzianum strain CI30 6 formulated in invert emulsion (water-in oil type) and applied
against other types of infections on fruits and other plant parts [15]-[17]. It is also important to mention that the
ingredients of our formulation of invert emulsion used in biopesticide have no environmental side-effects or
harmful toxic effects on treated fruits since they are mainly composed of natural substances that are used as food
additives or for manufacturi ng of cosmeti cs. Moreover, the fun gus strains of T. harzianum use d in biopes ticide are
easy to isolate from nature as a soil borne fungus and easy to cultivate on culture medium, in addition to that they
are cheap to maintain and easy to apply.
It is well-known that the various strains of T. harzianum can establish a mycoparasitic r elationship with their
host path ogens du ring parasi tism . This relat ionship coul d be done by interfe ring with t he developm ent and growth
of pathogens after conidial germination through coiling of their mycelium over the pathogen mycelium or by
disrupting the host fungus cell wall and consequent death of the pathogen [18] [19]. Antibiosis is another an-
tagonistic relationship made by certain strains of T. ha rzianum. This antibiosis could be done by producing an-
tifungal antibiotics in form of extracellular products (for example , hydrolyt i c e nz ymes like chitinases, glucanases

Y. A. Batta
795
and proteases) [18] [20]-[24]. Also, these strains could be propagated after successful host infection and distri-
bution wi thin t he cont aminat ed envir onment (horizont al tra nsmi ssion is m ost pr obable). The ab ove char acteri stics
make the se antago nistic f ungi excel lent can didates a s biocont rol agent s in the i ntegrate d managem ent stra tegies of
many plant diseases including postharvest diseases [21] [25].
From a practical point of view, formulation of T. harzian um strains is very necessary for protecting th e intro-
duced f ungus and e n hancing its effi c a c y . Until pre sent, many resea rchers h a ve a t tempted to formulate strains and
isolates of T. harzianum usi ng app rop riat e form ul ati ons. F or exa m ple, f orm ulat ion of T39 i sol ate of T. harzia num
in a powder form (commercially sold as TrichodexTR) then application of it against pathogens of many foliar
diseases such as Botrytis cinerea, Pseud oper onos pora cube nsi s, Sclerot ini a scl er otior um and Sphaerot hec a fus ca
(syn. S. fuliginea) in cucumber under commercial greenhouse conditions [26]. Also, formulation of the strain
1295-22 (T-22) of T. harzianum in a l iquid form (commercialized as T-22 Planter Box™) containing the conidial
suspension of the fungus used for the seed treatment against damping-off diseases [7] [27]. The same strain was
also formulated in a liquid form but this form contains the entire thallus of the fungus colonized on clay particles
(commercialized as RootShield™) for control of soil borne diseases [7] [27]. There is also another T. harzianum
product available in the Czech Republic and Denmark for the greenhouse use called commercially (Supresvit™)
in a dispersible powder containing conidia of the strain PV5736-89 and used for soil or potting mixes to control
damping-off a nd root r ots of ornam entals and forest t ree-seedlings [7]. In com parison wi th the above formul ations
of T. harzianum, our formulation of invert emulsion (water-in-oil type) contains the suspended conidia of the
fungus strain “TrichoPAL1” in the emulsion. These conidia were incorporated in the emulsion during its prepa-
ration so that the viability of these conidia could be preserved for long time realizing a significant reduction in
the mean of disease lesion diameter of pathogens challenged. In a previous research, one of th e prepared formu-
lations of invert emulsion (water-in-oil type) that contained the conidia of an effective strain of the fungus
“CI306” did not significa ntly lose its viability after 6 weeks of the ir introduction into emulsion. Also, it achieved
a high biological efficacy against B. cinerea on strawberry by decreasing the disease lesion diameter of the pa-
thogen by 44.0% to 55.0% compared to the control [15]. This proves again the benefits of our formulation and
its efficacy compared to oth er formulations.
5. Conclusion
An effective strain of T. harzianum was successfully formulated as biopesticide using a stable and non-viscous
formulation of invert emulsion (water-in-oil type) then tested against target pathogens of postharvest mold in-
fections on fruits of apple and pear. Treatments with the tested biopesticide have reduced significantly the mean
of disease lesion diameter on previously infected fruits before treatment. Also, they have protected fruits for a
long time from later infections (protection period up to 2.5 months). Due to above demonstration of biopesticide
effectiveness, it is recommended to test it at a large scale for the control of mold infections on marketed fruits
stored un der varia bl e c onditions .
Acknowledgements
I would like to thank the “Union of Arab Universities for Supporting the Palestinian Universities” for its fina n-
cial support of the present research project. My special thanks for the deanship of the scientific research of
An-Najah National University for their help in accomplish men t of this research project.
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