Biological Control 91 (2015) 88–93

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Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Biological control of anthracnose by postharvest application

of Trichoderma spp. on maradol papaya fruit
Nadia Landero Valenzuela a, Daniel Nieto Angel a,⇑, Daniel Téliz Ortiz a, Raquel Alatorre Rosas a,
Carlos Fredy Ortíz García b, Mario Orozco Santos c
a
Colegio de Postgraduados Campus Montecillo, Carretera México Texcoco Km. 6.5, Montecillo, Texcoco 56230, Estado de México, Mexico
b
Colegio de Postgraduados, Campus Tabasco. Periférico Carlos A. Molina, s/n, Carretera Cárdenas-Huimanguillo, km 3, Cárdenas, Tabasco, Mexico
c
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Carretera Colima-Manzanillo, km. 35, Colonia Predio La Escondida, Tecomán, C.P. 28930, Colima,
Colima, Mexico

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Trichoderma species against

Colletotrichum gloeosporioides on
papaya fruits.

The best strains in vitro were
Trichoderma longibrachiatum and
Trichoderma harzianum.

All tested strains of Trichoderma
showed mycoparasitism towards the
pathogen.

T. asperellum strain 1 and T. viride
were most effective in vivo.

There was no phytotoxicity by any of
the tested treatments.

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

Article history: Papaya is one of the most cultivated fruit in tropical and subtropical countries. It is affected by several
Received 10 June 2014 postharvest pathogens, including Colletotrichum gloeosporioides. The main goal of this research was to eval-
Revised 5 August 2015 uate the antagonistic capacity of five strains of Trichoderma against C. gloeosporioides using in vitro and
Accepted 18 August 2015
in vivo tests. All strains of Trichoderma inhibited radial growth on a plate of Colletotrichum by 50–60%.
Available online 18 August 2015
Moreover, Trichoderma longibrachiatum showed the highest colonization (87.45%) on Colletotrichum. In
tests to determine the mechanism of action, mycoparasitism was observed. Trichoderma harzianum was
Keywords:
mainly found invading mycelium of C. gloeosporioides. The severity measure showed that it was the
Trichoderma spp.
Colletotrichum gloeosporioides
interaction of the strain with the different time of inoculation that influenced the size of the lesion, with
Colonization the largest decrease in lesion size occurring when Trichoderma viride was inoculated 24 h before the
Inhibition pathogen. On the other hand, the Trichoderma strains did not cause color changes on papaya fruits.
Phytotoxicity Ó 2015 Elsevier Inc. All rights reserved.
Carica papaya

1. Introduction Fusarium spp. (Benato et al., 2001; Snowdon, 1990). Colletotrichum

The papaya fruit (Carica papaya) is very susceptible to posthar-
vest diseases by fungal pathogens such as Phoma caricae-papayae,
Rhizopus stolonifer, Alternaria alternata, Lasiodiplodia theobromae,
⇑ Corresponding author.
E-mail address: dnieto@colpos.mx (D. Nieto Angel).
gloeosporioides, the causal agent of anthracnose, is the most com-
mon postharvest papaya disease (Macedo, 2004; Cappellini et al.,
1998; Guzmán, 1998; Dickman and Alvarez, 1983; Arias, 1992).
Anthracnose can cause crop losses varying from 1 to 93% (Paul
et al., 1997) and is controlled with fungicides such as Benomyl
and Thiabendazole (Sanders et al., 2000; Tavares and e Souza,
2005; Liberato and y Tatagiba, 2001).

http://dx.doi.org/10.1016/j.biocontrol.2015.08.002
1049-9644/Ó 2015 Elsevier Inc. All rights reserved.
 N. Landero Valenzuela et al. / Biological Control 91 (2015) 88–93
The intensification of crop production in many low and middle
income countries is often accompanied by problems of pesticide
overuse and misuse, causing public health risk (Schreinemachers
and Tipraqsa, 2012). In spite of these problems, pesticides are
essential to ensure the food supply for the ever growing world pop-
ulation (Wang and Liu, 2007). In 2000, global pesticide use reached
3 million tons of active ingredients (Tilman et al., 2002). Brazil is
the third largest consumer of pesticides worldwide. In 2001, 300
million tons of formulated products were applied in Brazil and it
was ranked eighth in the world in terms pesticides used per
hectare (Fairbanks, 2001). Five thousand cases of poisoning are
officially reported annually in Brazil due to exposure to pesticides
(SINITOX, 2004). The lack of an effective regulation system, defi-
ciencies in product labeling, illiteracy, insufficient knowledge
about the risks, as well as the high cost of personal protective
equipment, are the causes of the high rate of exposure to pesticides
in developing countries (Maumbe and Swinton, 2003). With these
underlying problems, different alternatives to control diseases
have been developed to reduce the risks to human health, environ-
mental pollution, and to reduce the risk of hazardous chemical
wastes (Coates and Gowanlock, 1993, Prusky et al., 1999, Stevens
et al., 1997, Spadaro and Gullino, 2004). Biological control of fruit
diseases has shown promise in recent years. Antagonists have been
employed in grapes (Bulit and LaFont, 1972; Dubos, 1984), straw-
berry (Tronsmo and Dennis, 1977; Guédez et al., 2009), pineapple
(Lim and Rohrbach, 1980), citrus (Singh, 1980), apples and bananas
(Golam and Ilag, 1999). The fungus Trichoderma is a widely used
organism for biological control, and are active ingredients in com-
mercial products. The mechanisms responsible for the antimicro-
bial activity of Trichoderma include parasitism and competition
for space and nutrients (Sivakumar, 2001). Trichoderma is capable
of synthesizing different compounds such as proteins, enzymes,
and antibiotics, which increase its capacity to control phy-
topathogenic fungi (Al-Taweil et al., 2009). Recent studies have
shown that Trichoderma spp. secretes large lysis enzymes, such
as cellulases, chitinases and glucanases, during the mycoparasitism
process, that are capable of degrading the cell wall of the host fungi
and the genes that encode for these enzymes are activated in the
presence of mycelium or filtered culture medium of phy-
topathogenic fungi (Vinale et al., 2008). Given the successful use
of Trichoderma in other systems, the objective of the present study
was to evaluate the effect of five species of Trichoderma on
C. gloeosporioides (Penz.) Penz. and Sacc, the causal agent of
anthracnose on papaya, using in vitro and in vivo tests.
2. Materials and methods
2.1. Trichoderma isolates
Trichoderma viride Pers., Trichoderma longibrachiatum Rifai,
Trichoderma asperellum Samuels, Lieckfeld & Nirenberg, Tricho-
derma harzianum Rifai species were provided by CEPLAC (Comissa
o Executiva do Plano da Lavoura Cacaueira, for its acronym in Por-
tuguese) in Brazil. The isolates were cultured on Potato Dextrose
Agar (PDA) medium at room temperature (24 ± 2 °C). When the
typical salmon sporulation was observed, fungi spores were pre-
served in the ultra freezer at a temperature of 20 °C. Also, a
molecular identification was carried out with the purpose of con-
firming the species.
2.2. Pathogen isolates and pathogenicity tests
Maradol papaya (C. papaya) fruits with anthracnose symptoms
were collected from the Central Supply of Mexico City, Mexico. In
89
the laboratory, fragments of diseased tissue (composed of 20%
infected tissue and 80% healthy tissue) were excised
(5  5 mm) from lesions edges, were sampled and sown on potato
dextrose agar (PDA) plates (Cedeño et al., 1993; Dhingra and
Sinclair, 1995). Plates were incubated at room temperature
(26 ± 2 °C) for 10 days. A portion of mycelial tissue was transferred
to a fresh PDA plate in order to obtain a pure culture. Once the
identity of C. gloeosporioides was confirmed using taxonomic keys
(Sutton, 1992), seven strains (PVC28, PVC20, PVC16, PVC36, OC5,
OC8 and OC14) were identified. These strains were inoculated on
healthy papaya fruits with artificial wounds, using sterile wooden
sticks, mycelial agar plugs were placed on the wound, to reproduce
symptoms and re-isolate the causal agent (C. gloeosporioides) in
order to fulfill Koch’s postulates. Control fruits were inoculated
with PDA without the fungus. In addition to the use of morpholog-
ical keys, the identification of the pathogen was confirmed through
the molecular analysis for which a DNA extraction kit (DNeasyÒ
Quiagen) was employed, using generic primers (ITS2 and ITS5),
whose products were amplified by PCR and separated in
agarose gel, and finally they were sent to sequencing. The
ITS-5.8s-regions were amplified by PCR; PCR reactions were
performed in 25 lL reaction volumes and amplification was per-
formed in a thermocycler Touch C-1000 (BIO-RAD, Hercules, CA,
USA). The PCR products obtained were observed by agarose gel
electrophoresis 1%, stained with ethidium bromide (0.2 lg/lL).
The sequences were compared to those in the Gen-bank database.
The most pathogenic strain, which caused the most damage (OC14)
was selected for further experiments.
2.3. Antagonistic activity of Trichoderma spp. against
C. gloeosporioides in vitro
2.3.1. Trichoderma colonization on Colletotrichum
The dual cultures technique was used to test the antagonistic
activity of Trichoderma spp. (Sonnenbichler et al., 1983). A 5 mm
diameter disc containing active mycelium from C. gloeosporioides
was taken from the periphery of fungal colonies after 10 days of
growth. This was deposited towards the edge of the Petri dish. A
5 mm diameter Trichoderma spp. disc was taken from colonies after
5 days of growth, was deposited at the opposite end of the Petri
plate. The samples were then incubated at 26 °C ± 2 °C (Guédez
et al., 2009). Each strain was a treatment. The experiment was con-
ducted using a completely randomized design, it was established
in three times with a 24-h difference between. The data presented
were mean colony diameter. Superior values of colonization (50%)
were considered effective.
Eq. (1) was used to obtain the colonization percentage of
Trichoderma spp. on C. gloeosporioides:
DTP
C¼ ð100Þ ð1Þ
DE
where C is Trichoderma colonization percentage over Colletotrichum,
DTP is the distance travelled by the Trichoderma spp. colony on the
pathogen colony along the axis that separates both colonies and DE
is the distance between the two agar plugs whit fungus (4 cm)
(Rollan et al., 1999).
2.3.2. Inhibitory effect
The inhibitory effect of Trichoderma against Colletotrichum was
evaluated as the percentage inhibition calculated by the difference
between the growth of the pathogen confronted with the antago-
nist strain and the growth of the pathogen without antagonist
(control). The data were taken every 24 h for ten days (when the
control Petri dishes were covered).
90 N. Landero Valenzuela et al. / Biological Control 91 (2015) 88–93

2.3.3. Mechanism of action of Trichoderma on C. gloeosporioides
The Riddel micro culture technique described by Paul (1999)
was used to study the interhyphal interactions between each of
the five Trichoderma strains and C. gloeosporioides. This technique
consists of placing both fungi on opposite ends of the same thin
film of PDA on a glass slide that is supported by a glass rod in a
‘‘V” shape inside a Petri dish.
After placing mycelium of each fungus on the PDA (with a 24-h
difference between C. gloeosporioides and Trichoderma, placing first
to the pathogen due its slow growth), a sterile coverslip was placed
on the thin film. To maintain humidity inside the Petri dish, 10 mL
of 10% glycerol was added.
When the agar disc was covered with growth of both fungi,
glycerol was replaced by 10% formaldehyde for 24 h to fix the fun-
gal structures. The microscope slide was then removed from the
Petri dish to prepare two smears. The coverslip was placed in
another clean glass slide with a drop of lactophenol cotton blue.
The next step consisted of removing the agar disc from the original
microscope slide, placing a drop of lactophenol cotton blue and a
clean coverslip, and thus two smears were obtained. When the col-
oring excess was eliminated until the mycelium of both fungi were
clearly observed, the preparations were sealed whit a nitrocellu-
lose–acetone mixture and tested microscopically to determine
the mechanism of action employed by Trichoderma strains. The
experiment was conducted for four days, until that the agar disc
was covered with growth of both fungi. Subsequently was
observed if there were vacuolization, penetration and adherence.
digitization of the pictures a Kodak camera Z712 IS OF 7.1 mega-
pixel was employed. The photographs were taken at 1024  768
megapixels at a distance of 1 m, illuminated with a 20 W fluores-
cent lamp. The lens aperture and shutter speed were maintained
at f/2.8 and 1/30 s, respectively. The angle between the camera lens
and the source of illumination was approximately 45°. Color red,
green and blue parameters were obtained using Photoshop soft-
ware, which were then statistically analyzed (Lúquez and
Aguilera, 2005).
2.5. Statistical analysis
The experiment used a completely randomized design with 12
replicates (4 per fruit). The experiments were established three
times, with 24 h between each experiment. The data presented
here is an average. There were 17 experimental treatments
(Table 1) in vivo, consisting of each of the 5 strains with three repli-
cates, as well as two controls, Imazalil (N 1-[2-2,4-dichlorophene
thyl)-2-(2propeniloxi) etil]-1H-imidazol) control and absolute con-
trol (untreated fruits).
The data obtained was subjected to normality (Shapiro Wilk)
and homoscedasticity (Barret) tests, transforming them when it
p
was needed ( 2). Analysis of variance and multiple mean separa-
tion tests (Tukey, P = 0.05) were carried out on the data. Area under
the disease curve was calculated for in vivo experiments.
3. Results and discussion

2.4. Antagonistic activity of Trichoderma spp. on C. gloeosporioides 3.1. Antagonistic activity of Trichoderma spp. on C. gloeosporioides
in vivo in vitro

2.4.1. Effectiveness of Trichoderma spp. against C. gloeosporioides Measures of growth of each of the Trichoderma strain were car-
lesions on postharvest papaya fruits ried out during the first 10 days of incubation, during which time C.
Maradol papaya fruits were obtained from a producer in the gloeosporioides completely covered the Petri dish (Fig. 1). All strains
state of Oaxaca, México. Healthy fruits with no physical damage showed superior colonization than the threshold value of 50%
or apparent signs or symptoms of the disease were selected. Each colonization. The isolate with the lowest level of colonization
fruit was disinfected with 2% sodium hypochlorite for 3 min, while was T. asperellum strain 2 (7.8043). This species was previously
rubbing the surface gently to avoid damaging the epidermis; then reported as an antagonistic of pathogens in vitro, stopping the
the fruits were rinsed with sterile distilled water and left to dry at mycelial growth of Pythium myriotylum Drechsler (Mbarga et al.,
room temperature for 20 min on blotting paper. 2012). T. longibrachiatum showed the highest colonization value
The effectiveness of the different species of Trichoderma to sup- (9.3520) (Table 2). It was observed at the end of the test, that
press development of lesions was tested by adding the bio-control T. longibrachiatum did not cover the colony of Colletotrichum
agent at three time points: (1) 24 h before the pathogen, (2) at the
same times as the pathogen and (3) 24 h after the pathogen was
inoculated onto fruit. Trichoderma was inoculated to the artificial Table 1
Treatments used to evaluate the effect of the different Trichoderma species as
lesions (each of 2 mm depth and 2 mm diameter) made with ster-
biological control of C. gloeosporioides on papaya fruits.
ilized wooden chopsticks. Each strain of Trichoderma spp. (20 lL)
was added at a concentration of 1  106 spores/mL (20,000 spor- Treatments Repetitions
es/20 lL) per lesion (4 lesions per fruit) using a micropipette). A T1C10 12
drop of similar size of the pathogen was also applied to the com- T1C8 12
T1C7 12
mercial control (Imazalil (N 1-[2-2,4-dichlorophenethyl)-2-(2pro
T1C5 12
peniloxi) etil]-1H-imidazol) and absolute control (untreated T1C1 12
fruits). The pathogen was inoculated to the lesions according to T2C10 12
the times mentioned previously for Trichoderma spp. The treat- T2C8 12
ments were incubated in wet chambers (sealed plastic containers), T2C7 12
T2C5 12
with a separate container for each fruit, at 26 ± 2 °C and with a rel-
T2C1 12
ative humidity of 95%. T3C10 12
T3C8 12
2.4.2. Severity testing on papaya fruits T3C7 12
T3C5 12
The equatorial diameter of the lesion caused by the pathogen
T3C1 12
was measured every 24 h after inoculation. Absolute control 12
Imazalil 12
2.4.3. Phytotoxicity testing on papaya fruits
T1 = 24 h before the pathogen, T2 = at the same time as the pathogen, T3 = 24 h
Phytotoxicity was assessed through the color changes in treated after the pathogen, C1 = Trichoderma viride, C5 = Trichoderma longibrachiatum,
fruits in relation to the absolute control (untreated fruits), using C7 = Trichoderma asperellum strain 1, C8 = Trichoderma asperellum strain 2,
digital pictures of the treated fruits with different strains. For C10 = Trichoderma harzianum.
 N. Landero Valenzuela et al. / Biological Control 91 (2015) 88–93
Fig. 1. Antagonism test of (A) Trichoderma viride, (B) Trichoderma longibrachiatum, (C) Trichoderma asperellum strain 1, (D) Trichoderma asperellum strain 2, (E) Trichoderma
harzianum on Colletotrichum gloeosporioides and (F) Colletotrichum gloeosporioides. Arrows indicate the growth of Trichoderma.
completely (Fig. 1B), in contrast to the rest of strains that covered
the surface of C. gloeosporioides fully (Fig. 1A, C, D and E). This sug-
gests that T. longibrachiatum had a faster growth rate than the
other species, but when in contact with the pathogen its growth
slowed. T. harzianum has been reported as a biopesticide of Botry-
odiplodia theobromae Pat. and C. gloeosporioides in vitro
(Wijeratnam et al., 2008). Guédez et al. (2009) showed that this
fungus is considered an effective biological control agent against
postharvest diseases of strawberry because of its rapid growth
and capacity to reduce the growth of R. stolonifer Ehrenb. (Ex Fr.)
Lind, Mucor spp., Penicillium digitatum (Pers.:Fr.) Sacc., Rhizoctonia
solani Kühn, Aspergillus niger Van Tieghem and Pythium spp. in
strawberry fruits.
T. asperellum strains 1 and 2 showed similar behavior when
growing alongside pathogen (P < 0.05). The morphology of these
strains showed the same characteristics (Fig. 1).
C. gloeosporioides inhibition by the Trichoderma strains in dual
cultures did not show statistically significant differences (Table 3).
Inhibition was higher than 50% for each of the studied strains, with
T. longibrachiatum showing the highest inhibition and T. harzianum
the lowest. The opposite results were reported by Golam and Ilag
(1999) who obtained highest inhibition for T. harzianum followed
by T. viride. Sousa-Rocha and Oliveira (1998) found C. gloeospori-
oides was 100% inhibited by Trichoderma koningii Oudem.
T. longibrachiatum was confirmed as the species with the high-
est effectiveness value on C. gloeosporioides in vitro (Tables 2 and 3).
The criteria to select the best strains took into consideration the
highest colonization and inhibition percentage.
Antagonism was verified on dual micro-cultures by observation
under a compound microscope, considering vacuolization, invasion
and adherence (Fig. 2). It was observed that each of the
Trichoderma strains showed characteristics of mycoparasitism
Table 2
Percentage of colonization of Trichoderma spp. on Colletotrichum gloeosporioides
in vitro.
Treatment Colonization*
Trichoderma longibrachiatum 9.3520a**
Trichoderma harzianum 9.1195a
Trichoderma viride 9.1174a
Trichoderma asperellum strain 1 7.9695b
Trichoderma asperellum strain 2 7.8043b
p
*
Processed data is shown with 2.
**
Means followed by the same letter are statistically the same (Tukey 0.05).
91
Table 3
Percentage of inhibition of Colletotrichum gloeosporioides by Trichoderma spp. in vitro.
Treatment Inhibition (%)*
Colletotrichum-T. longibrachiatum 55.820a
Colletotrichum-T. viride 55.416a
Colletotrichum-T. asperellum strain2 55.312a
Colletotrichum-T. asperellum strain1 55.289a
Colletotrichum-T. harzianum 53.264a
Control 0.00b
*
Means followed by the same letter are statistically the same (Tukey 0.05).
towards the pathogen fungal mycelium, especially T. harzianum
which caused the most drastic damage, showing invasion of the
C. gloeosporioides mycelium (Fig. 2F). Elad (2000) confirmed that
among the mechanisms of action of this species on Botrytis cinerea
Pers.:Fr. were competition, restriction of enzyme production by the
pathogen and induced resistance. The remaining antagonists were
parallel attached to the pathogen mycelium, causing rupture of
membranes, as a result vacuolization in the different sections of
the hyphae, considering dead this tissue. Golam and Ilag (1999)
state that T. harzianum and T. viride are parallel adhered too, in
addition to rolling around the L. theobromae. (Syn. B. theobromae
Patouillard).
3.2. Antagonistic activity of Trichoderma spp. against C.
gloeosporioides in vivo
Although none of the Trichoderma spp. could completely inhibit
lesion development on papaya fruits, they significantly reduced the
lesions artificially inoculated at three different times (T1 = 24 h
before pathogen inoculation, T2 = at the same time as the patho-
gen, and T3 = 24 h after pathogen inoculation). Similar results were
reported by Golam and Ilag (1999) that T. viride and T. harzianum
could not fully control the growth of L. theobromae on banana fruits
but there was a reduction of up to 65.06% by T. viride.
The reduction in lesion diameter was more effective when
T. viride was inoculated 24 h before inoculating the pathogen,
while the highest reduction in diameter was obtained when
T. longibrachiatum was inoculated 24 h after C. gloeosporioides.
Significant statistical differences mainly occurred between the
T1C1 and T2C7 treatments with respect to the absolute control
(untreated fruits). Other authors have found that better results
were obtained when inoculating the antagonist prior to pathogen
92 N. Landero Valenzuela et al. / Biological Control 91 (2015) 88–93

Fig. 2. Colletotrichum gloeosporioides hyphae interacting with (A) Trichoderma viride, (B) Trichoderma longibrachiatum, (C) Trichoderma asperellum, strain 1, (D) Trichoderma
asperellum strain 2, E y (F) Trichoderma harzianum. Red arrows indicate Colletotrichum hyphae, black arrows indicate Trichoderma hyphae. (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article.)

application compared to application simultaneously or after the Table 4

pathogen (Golam and Ilag, 1999). Effect of Trichoderma strains on the growth of the lesions caused by Colletotrichum
gloeosporioides on papaya fruit.
It was observed that the lesion value was independent of the
strain or the inoculation time, but reflected the interaction Treatment Application Severity mean* Lesión diameter
between them, as shown for the last two treatments (when time (mm)
T. asperellum strain 1 was inoculated at the same time as the patho- Absolute control 46.436 a
gen, and when T. viride was inoculated 24 h before the pathogen) Trichoderma T3 37.900 ba
(Table 4), in which neither the strain nor the inoculation time were longibrachiatum
Trichoerma harzianum T2 32.170 bac
the same. This was also the case for the other treatments where Imazalil 31.656 bac
there was no significant statistical difference, in contrast to T1C8, Trichoderma asperellum T2 31.390 bdac
T2C8 and T3C8 treatments where there appeared to be an influ- Cepa 2
ence of T. asperellum strain 2 on the growth of the lesion. Trichoderma asperellum T1 24.665 bdec
Cepa 2
This antagonist has been studied against Thielaviopsis paradoxa
Trichoderma asperellum T3 23.865 bdec
(De Seynes Hohn) in pineapple, where it effectively controlled Cepa 2
pathogen growth, having a fungicidal effect (Wijesinghe et al., Trichoderma viride T3 23.284 bdec
2010). The antagonist was not found to be effective in this study, Trichoderma harzianum T3 22.150 dec
where the treatments mentioned previously (T1C8, T2C8 and Trichoderma asperellum T1 20.990 dec
Cepa 1
T3C8) equaled the absolute control (untreated fruits). Trichoderma harzianum T1 20.628 dec
Díaz and Vila (1990) found no reduction in lesion growth when Trichoderma asperellum T3 20.305 dec
T. viride and P. digitatum were inoculated simultaneously on Cepa 1
oranges, as the inoculation time of the pathogen increased, the Trichoderma T2 19.115 dec
longibrachiatum
lesions were reduced (Díaz and Vila, 1990); whereas in the present
Trichoderma viride T2 18.196 dec
study there was no difference in lesion growth when the Trichoderma T1 16.248 de
antagonist was inoculated before, simultaneously or after the longibrachiatum
pathogen in all treatments (Table 4). The highest reduction of Trichoderma asperellum T2 12.223 e
lesion growth occurred when T. viride was inoculated 24 h before Cepa 1
Trichoderma viride T1 10.495 e
C. gloeosporioides (77.40%), while the lowest reduction occurred
when T. longibrachiatum was applied 24 h after the pathogen T1 = 24 h before the pathogen, T2 = at the same time as the pathogen, T3 = 24 h
(18.38%). This suggests that T. viride as bio-pesticide must be after the pathogen, C1 = Trichoderma viride, C5 = Trichoderma longibrachiatum,
C7 = Trichoderma asperellum strain 1, C8 = Trichoderma asperellum strain 2,
applied pre-harvest to reduce loss during transportation and sale.
C10 = Trichoderma harzianum.
Piotrowska and Dorszewski (1996) report T. viride as one of the *
Means followed by the same letter are statistically the same (Tukey 0.05).
most effective antagonists against Alternaria alternata on potato,
improved upon only by T. koningii. The effectiveness of T. viride
was confirmed by Nallathambi et al. (2009) who isolated the found that Trichosetin produced in dual culture by T. harzianum
superior strain T. viride CIAH240. with Catharanthus roseus L. inhibited the growth of roots in
Considering that the value obtained with the fruits used as the different plant species and that cells of roots treated with this
absolute control (untreated fruits) represents the appropriate metabolite died.
coloring for papaya maradol variety, there was no significant sta-
tistical difference with the different inoculated strains. There was
a significant difference between the effects of T. viride and 4. Conclusions
T. asperellum strain 1 (P = 0.0021), but there was no difference
between these treatments and the absolute control (untreated The strains with the highest colonization on C. gloeosporioides
fruits). The commercial fungicide (Imazalil) did not cause in vitro were T. longibrachiatum and T. harzianum, while there
phytotoxicity (assessment of color) on papaya fruits (Table 5). was no significant statistical difference between treatments for
Contrasting results were reported by Marfori et al. (2003) who inhibition. All of the antagonistic strains showed mycoparasitism
 N. Landero Valenzuela et al. / Biological Control 91 (2015) 88–93
Table 5
Phytotoxicity test of Trichoderma on postharvest papaya fruits.
Treatments Means of RGB
Trichoderma viride 147.631 a*
Absolute control 133.694 ba
Trichoderma harzianum 131.215 ba
Trichoderma asperellum strain 2 124.234 ba
Trichoderma longibrachiatum 123.446 ba
Imazalil 121.955 ba
Trichoderma asperellum strain 1 115.436 b biocontrol agent for Pythium myriotylum causal agent of cocoyam (Xanthosoma
*
Means followed by the same letters are statistically the same (Tukey 0.05).
as the mechanism of action towards the pathogen. For the in vivo
treatments, time 0 h-T. asperellum strain 1 and time 24 h before
T. viride were the most effective treatments, with the highest
efficiency of 77.40%. There was no significant difference in
phytotoxicity for any of the tested treatments.
Acknowledgments
Special thanks to Dr. Cristian Diaz Nava, for their selfless sup-
port in reviewing this article.
Special thanks to Colegio de Postgraduados Trust for their finan-
cial support to complete this work.
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