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Trichoderma from Aceh Sumatra reduce Phytophthora lesions on pods
and cacao seedlings
Rina Sriwati
a
, Rachel L. Melnick
b,
⇑
, Rizky Muarif
a
, Mary D. Strem
b
, Gary J. Samuels
c,d
, Bryan A. Bailey
b
a
Agrotechnology Department, College of Agriculture, Syiah Kuala University, Banda Aceh, Indonesia
b
USDA-ARS Sustainable Perennial Crops Lab, Beltsville, MD, USA
c
USDA National Institute of Food and Agriculture, Washington, DC, USA
d
USDA-ARS Systematic Mycology and Microbiology Lab, Beltsville, MD, USA
highlights
Trichoderma spp. are endophytes of
Indonesian cacao leaves and roots.
T. virens isolate Tv was a
mycoparasite and suppressed P.
tropicalis and P. palmivora.
T. virens isolate Tv reduced P.
palmivora lesion expansion on
detached cacao pods.
T. virens isolate Tv reduced P.
palmivora seedling leaf lesions on
cacao.
graphical abstract
Isolate
Trichoderma spp.
from cocoa ssue
Idenfy
species
Lab assays
determined
isolate T.
virens Tv is
mostly likely
biological
control agent
T. virensTv reduced P.palmivora
lesion size in detached pod assay
Control
T. virensTv
T. virensTv reduced P. palmivora
lesion incidence on seedlings
Cntl Tv
Dual Plate
assay
Anbiosis
Mycoparasism
article info
Article history:
Received 29 July 2014
Accepted 16 April 2015
Available online 12 May 2015
Keywords:
Cacao
Black pod rot
Trichoderma
Phytophthora
abstract
The cocoa tree, Theobroma cacao L., suffers large yield losses in Aceh Indonesia due to the disease black
pod rot, caused by Phytophthora spp. Despite having the largest area under cacao production in Sumatra,
farmers in the Aceh region have low overall production because of losses to insect pests and black pod
rot. Trichoderma spp. were isolated from the roots and leaves of cacao trees and screened as potential bio-
logical control agents. Isolates used in the study were Trichoderma asperellum isolates T2 and T4,
Trichoderma longibrachiatum isolates T15 and T16, and Trichoderma virens isolates T1 and Tv. T1, T2,
T4, and Tv completely colonized and destroyed Phytophthora tropicalis and Phytophthora palmivora myce-
lium in precolonized plate assays. All six isolates reduced P.tropicalis, but none reduced the growth of P.
palmivora in dual plate assays. Phytophthora growth was suppressed on MIN media amended with sterile
heat inactivated Trichoderma culture filtrates, with Tv best suppressing growth of both Phytophthora spp.
T. virens isolate Tv was the only isolate observed coiling around P.tropicalis mycelium and disrupted the
formation of P. palmivora sporangia. Of all six isolates, only Tv reduced P.palmivora lesion expansion in a
detached pod assay, reducing severity by 71%. Tv also reduced P. palmivora infection on seedlings when
applied aerially at 1 10
6
and 1 10
8
conidia/ml, by 19% and 59%, respectively. T. virens isolate Tv is a
mycoparasite, antagonizes Phytophthora in a dual plate assay, and shows antibiosis against
Phytophthora spp., suggesting that multiple modes of action contribute to its ability to limit
Phytophthora lesion expansion on cacao pods and seedlings.
Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.biocontrol.2015.04.018
1049-9644/Published by Elsevier Inc.
⇑
Corresponding author at: USDA National Institute of Food and Agriculture, 800
9th Street, SW, Washington, DC 20024, USA. Fax: +1 202 401 4980.
E-mail address: rachelmelnick@gmail.com (R.L. Melnick).
Biological Control 89 (2015) 33–41
Contents lists available at ScienceDirect
Biological Control
journal homepage: www.elsevier.com/locate/ybcon
1. Introduction
Indonesia has been the world’s third largest cocoa producers
since the 1980s. Theobroma cacao L. (cacao) is an understory crop
currently grown over a large area in the Aceh Province of
Indonesia. The area under cacao production is significantly higher
in Aceh than the other provinces on Sumatra, yet overall yield is
well below the national average of 440 kg/ha/year because of lim-
iting factors such as nutritional deficiencies, insect pests, and dis-
eases (International Cocoa Organization, 2014). Black pod
disease, caused by the stramenophile Phytophthora palmivora,is
the most important cause for cacao yield loss in the region
(Purwantara et al., 2004), although Phytophthora citrophora
(Appiah et al., 2004) and Phytophthora tropicalis have also been
reported (Bowers et al., 2001). Approximately 70% of cacao pods
are lost to the disease black pod rot (Nasir personal communica-
tion, 2012). Roots, stems, flowers, and leaves can also be infected
by Phytophthora spp. (Guest, 2007). Farmers use of shade manage-
ment, phytosanitation, pesticides, and tolerant cultivars have had
limited impact on managing black pod rot (Bowers et al., 2001;
Ndoumbe-Nkeng et al., 2004). Pesticides can be effective, but are
costly and can pose a health risk for farmers.
Cacao trees are home to a diverse endophytic microbial com-
munity including Bacillus spp. (Melnick et al., 2011),
Colletotrichum spp. (Mejía et al., 2008; Rojas et al., 2010), and
Trichoderma spp. (Hanada et al., 2008, 2010; Samuels et al.,
2006b). Of these, Trichoderma isolates have been the focus for bio-
logical control of cacao diseases. Trichoderma spp. have increas-
ingly been found as endophytes in aerial plant tissue in tropical
plants (Chaverri et al., 2011; Gazis and Chaverri, 2010).
Trichoderma spp. have been isolated as endophytes from several
Theobroma spp. including Theobroma gileri (Evans et al., 2003;
Samuels and Ismaiel, 2009), Theobroma grandiflorum (Hanada
et al., 2010; Holmes et al., 2004), and T. cacao (Hanada et al.,
2008, 2010; Samuels and Ismaiel, 2009; Samuels et al., 2006b).
Trichoderma spp. have previously been shown to reduce the
growth of cacao pathogens and subsequent disease through
antibiosis, antagonism, mycoparasitism, and induced resistance.
Metabolites from endophytic Trichoderma asperellum and
Trichoderma harzianum reduced the growth of Moniliophthora ror-
eri, causal agent of frosty pod pathogen, while the fungi parasitized
M. roreri mycelium (Bailey et al., 2008). Mycoparasitic Trichoderma
stromaticum was found colonizing cacao tissue infected with
Moniliophthora perniciosa, causal agent of witches broom disease
(Samuels et al., 2000). T.stromaticum suppressed basidiocarp for-
mation (Samuels et al., 2000) and reduced the number of pods lost
to M. perniciosa (Medeiros et al., 2010). Additionally, endophytic
colonization of cacao seedlings with Trichoderma spp. induced
plant stress and defense-related genes (Bailey et al., 2006).
Trichoderma spp. have been successfully used in field trials to
manage black pod rot of cacao. Trichoderma martiale limited black
pod rot caused by P. palmivora in Brazil (Hanada et al., 2008, 2009).
Treatment of trees in Cameroon with T. asperellum reduced the
number of diseased pods (Tondje et al., 2007), while T. asperellum
strain PR11 reduced disease rates and incidence of black pod rot
caused by Phytophthora megakarya over three years (Deberdt
et al., 2008). Due to previous success utilizing Trichoderma to man-
age cacao diseases, we wanted to screen native endophytic
Indonesia Trichoderma spp. as potential biological control agents.
The cacao agroecosystem in Indonesia is a well-studied system,
but work has primarily focused on how managing cacao planta-
tions impacts biodiversity, not on managing cacao diseases.
Because of the concerns about the impact of pesticides on this bio-
diversity, utilizing a biological control agent consisting of a native
fungal isolate could reduce the negative impact of cacao farming
on native biodiversity. Additionally, increasing cacao production
after the 2004 Indian Ocean Tsunami is important to economic
recovery in the region. Native isolates were screened, as they could
be better adapted to the environment and microbial communities
occurring in the Indonesian cacao. Previous success utilizing
Trichoderma to manage cacao diseases led us to investigate
whether Trichoderma spp. could manage black pod rot on Aceh
cacao. The goals of this research were to (1) obtain and identify
Trichoderma spp. from cacao trees in the Aceh Province of
Indonesia, (2) determine the potential modes of action for antago-
nistic Trichoderma spp. against Phytophthora and (3) to test the
ability of the isolates to be biological control agents for both
Phytophthora seedling blight and black pod rot.
2. Materials and methods
2.1. Isolation and molecular identification of Trichoderma spp. from
cacao
Native Trichoderma isolates were obtained through three sam-
plings. For the first isolation, healthy cacao leaves and roots were
harvested from cocoa plantations in East Aceh, Langsa, and Aceh
Besar, in the Aceh Province of Indonesia. Tissue was transported
to the laboratory and processed within 4 h of collection. Sections
(2 cm 2 cm leaf, 2 cm long roots) were excised and surface ster-
ilized in 90% ethanol for five minutes, following by rinsing in sterile
distilled water six times. Afterward the tissue was further steril-
ized in 10% bleach for five minutes followed by rising tissue in ster-
ile distilled water five times. Tissue was plated into potato
dextrose agar (PDA, DC, Franklin Lakes, NJ) and incubated at room
temperature for up to a week. Hyphae growing from the tissue
were subcultured and those morphologically resembling
Trichoderma were maintained. Isolates T1, T2, T15, and T16 were
obtained using this method.
Isolate Tv was obtained through a separate method. For isolate
Tv, root tissue was surface sterilized in 10% bleach for five minutes
followed by rinsing tissue in sterile distilled water three times.
Tissue was plated as previously described. Isolate T4 was isolated
from rhizosphere soil from around the roots of a cacao trees. One
gram was dilution plated onto PDA. Plates were incubated and sub-
culturing was conducted as previous stated. Trichoderma isolates
were maintained on cornmeal dextrose agar (CMDA) and are stored
at the U.S. National Fungus Collection at USDA ARS in Beltsville, MD.
For species identification, DNA was isolated from 7-day-old
Trichoderma cultures growing in 100 ml of potato dextrose broth
(PDB) following the methods of Dodd et al., 2002. Portions of the
translation elongation factor alpha-1 (TEF1) gene were amplified
with primers EF1-728F and TEF1 rev following the protocol in
Samuels et al. (2006a). DNA sequences of both strands of the
TEF1 amplicons were sequenced using the BigDye Terminator cycle
sequencing kit (Applied Biosystems, Foster City, California) and
directly analyzed on an ABI Prism 3100 genetic analyzer (Applied
Biosystems). DNA sequences were aligned to known species in
the GenBank database using the MEGA 6 (Tamura et al., 2013).
2.2. Phytophthora source and maintenance
P. tropicalis isolate 73–74, collected by H. Purdy (Univ. of
Florida) from an infected cacao pod in Ecuador, was used in labo-
ratory screening of Trichoderma spp. P. palmivora used for labora-
tory assays and plant disease screenings was collected from
infected cacao pods at the experimental farm of Kaliwining by
the Indonesian Cocoa and Coffee Research Institute, Jember, Java,
Indonesia. Phytophthora isolates were maintained on V8-PARP
agar.
34 R. Sriwati et al. / Biological Control 89 (2015) 33–41
2.3. Screening of Trichoderma isolates for qualities of a biological
control agent
2.3.1. Precolonization plate assay for mycoparasitism
The pre-colonized plate assay was conducted following Bailey
et al. (2006) utilizing P. tropicalis and P. palmivora in separate
experiments. Six Trichoderma isolates, representing phylogeneti-
cally distinct species, were chosen to test as potential biological
control agents. Trichoderma isolates T1, T2, T4, T15, T16, and Tv
were used in all laboratory assays. A 0.5 6 cm strip of the P. trop-
icalis or P. palmivora was excised from a 7-day-old colony and
placed onto middle of a 9 cm V8 agar plate. Four replicate plates
were used per treatment. Plates were incubated at 25 °C in the
dark. After 7 days, the plates were fully colonized by
Phytophthora, and were inoculated with a 0.5 4 cm strip of each
Trichoderma isolate by place the strip perpendicularly across the
Phytophthora strip near one end. There were four replicate plates
per isolate. Plates were maintained at 25 °C in the dark and
observed daily for fungal growth. Seven days after inoculation with
Trichoderma, 3 mm plugs were removed from the plate at 0, 2, and
4 cm from the Trichoderma inoculation point and were placed on
6 cm Petri dishes of MIN media (Bailey et al., 2006). Plates were
incubated at 25 °C for 14 days and observed daily. The presence
of Trichoderma and the respective Phytophthora sp. were recorded.
The experiment was repeated twice.
2.3.2. Dual plate assay to test for antibiosis
A dual plate assay was performed to determine whether
Trichoderma spp. were antagonistic to P. tropicalis and P. palmivora
in separate experiments. A 5 mm plug of P. tropicalis or P. palmivora
was placed on one side of a 2% glucose CMDA plate and a 5 mm
plug of a Trichoderma isolate was placed on the opposite edge of
a 9 cm petri dish. Control plates consisted of P. tropicalis,P. palmi-
vora,orTrichoderma alone. Four replicate plates were used per
treatment. Colony diameter of both Trichoderma and
Phytophthora spp. was measured daily for 4 days and used to deter-
mine growth rate as (mm/day). The experiment was repeated
twice.
2.3.3. Mycoparasitism assay on glass slides
A 2.5 cm 3.0 cm area of a sterile glass slide in a Petri dish was
covered with 1.5% molten agar and allowed to cool. A 5 mm plug of
P. tropicalis or P. palmivora was placed on one side of the agar.
Slides were aseptically maintained in sterile 9 cm Petri dishes
and were incubated at 25 °C for 4 days, after which a 5 mm plug
of one of the Trichoderma isolates was placed on the opposite side
of the agar. Slides were incubated at 25 °C and hyphal growth was
observed daily for one week after inoculation under a dissecting
scope. Four replicate slides were used per treatment. After seven
days, slides were stained with lactophenol blue and observed
microscopically at 20and 100at the interaction zone between
P.tropicalis and the tested Trichoderma sp. Observations of
Trichoderma coiling around and/or penetrating Phytophthora and
abnormalities in the hyphal growth were noted. The experiment
was repeated three times.
2.3.4. Antibiosis assay using culture filtrates
An antibiosis assay was conducted following Bailey et al. (2006).
Flasks containing 100 ml of MIN broth (Srinivasan et al., 1992)
were inoculated with 1 ml of a 1 10
6
conidia/ml suspension of
the each Trichoderma species (from 7-day-old cultures on PDA)
and incubated on an orbital incubator at 25 °C and 110 rpm. Four
replicate flasks were used per treatment. After seven days, mycelia
were filtered from the growth medium using sterile cheese cloth
and funnels. Filtrates were incubated at 90 °C for two hours to
inactivate enzymes. Five milliliters of filtrates were added to 5 ml
of molten 1or 2MIN agars and were then poured into 6 cm
Petri dishes. Plates were inoculated with a 4 mm plug of P. tropi-
calis or P. palmivora from one-week old cultures. MIN plates were
incubated at 25 °C and radial growth was measured daily for one
week. Radial growth was calculated as mm/day. Inhibition of
Phytophthora mycelial growth was recorded as the difference
between mean radial growth in the presence and absence of the
fungal filtrate. The experiment was repeated twice.
2.4. Testing Trichoderma ability to reduce P. palmivora lesions on
detached cacao pods
Approximately 3-month-old cacao pods were harvested from
TSH clones from plantations in Blang Barou Village, Pidie Jaya,
Aceh, Indonesia. Before harvest, the plantation was inspected and
determined to be free of black pod rot. Pods were transported to
the laboratory. Trichoderma conidia were harvested for all isolates
from one-week old cultures grown on PDA by rinsing the plates
with sterile distilled water. Spores concentrations were deter-
mined using a hemocytometer and attenuated to 1 10
6
-
spores/ml. The suspension was sprayed on all surfaces of eight
replicate pods per isolate, so that the pods were completely wet-
ted, using a handheld aerosol sprayer. Pods were placed in a plastic
box at 28 °C for one day to maintain humidity. One day after appli-
cation of Trichoderma isolates, pods were inoculated with P. palmi-
vora as follows. The epidermis of each pod was wounded at three
points 1 cm apart using a 1 mm needle. The wounded areas were
covered with a 1 cm 1 cm agar square removed from a 7-day
old culture of P. palmivora grown on PDA. There were eight repli-
cate pods per treatment. Pods were incubated for 5 days at room
temperature in a 30 cm 30 cm plastic bag. Lesion diameter was
measured daily. Area under the disease progress curve (AUDPC)
was calculated to assess disease progression. The experiment was
repeated twice.
2.5. Testing ability of Trichoderma virens isolate Tv to reduce P.
palmivora lesions on seedlings
Based on results of laboratory studies and the detached pod
assay, T. virens isolate Tv was the best antagonist of Phytophthora
and was selected to evaluate the effective concentration of conidia
that would best reduce P. palmivora seedling blight. Seeds from
pods of similar size and age were obtained from a private planta-
tion in Baro Tunoung, Pidie Jaya district, Aceh, Indonesia. The muci-
lage and seed coat were removed and seeds were placed onto
wetted cotton for germination. After germinating on wet cotton
for five days, seedlings were planted into the center of 1 kg poli-
bags containing sterilized field soil and compost (2:1). Plants were
maintained in a randomized block design on a greenhouse bench.
Fifteen days after planting, T. virens isolate Tv conidial suspensions
were applied to six replicate plants for each concentration. Conidial
suspensions were prepared as previously described. Conidial con-
centration was adjusted in sterile water to 1 10
4
,110
6
, and
110
8
conidia/ml. Conidia were applied in the in the evening by
spraying all aerial portions of the plant with handheld aerosol
sprayers until the entire seedling was wet. Control plants were
sprayed with sterile water. After application of T. virens, plants
were returned to the greenhouse and covered with a clear plastic
tent for 24 h to maintain humidity.
Seven days after application of T. virens, the seedlings were
inoculated with P. palmivora. P. palmivora sporangia were obtained
from P. palmivora infected cocoa pods following the methodology
of Sriwati and Muarif (2012). The P. palmivora sporangia concen-
tration was adjusted to 1 10
6
sporangia/ml using a hemacytome-
ter. Despite efforts, no zoospores were induced or observed prior to
or after application. The spore suspension was sprayed onto
R. Sriwati et al. / Biological Control 89 (2015) 33–41 35
seedlings using a handheld aerosol sprayer. Plants were returned
to the greenhouse and covered a clear plastic tent for 24 h to main-
tain humidity. Two weeks after inoculation with P. palmivora,
plants were observed for overall disease incidence, the number of
infected leaves, and the total number of leaves. The experiments
were repeated twice.
2.6. Statistical analysis of data
Data were analyzed for statistical significance using analysis of
variance via PROC GLM in SAS 9.2 (SAS Institute, Raleigh, NC) fol-
lowed by Tukey post hoc testing with
a
= 0.05. Laboratory experi-
ments with P. tropicalis and P. palmivora were conducted and
analyzed independently. In all analyses, there were no statically
significant differences between repeated experiments; therefore,
data from replicate experiments was combined for statistical
analysis.
3. Results
3.1. Isolation and identification of endophytic Trichoderma species
Of the six unique isolates from Aceh, Indonesia, all were in the
Trichoderma longibrachiatum clade of Trichoderma. Isolates T1, T2,
T15, and T16 were isolated as leaf endophytes. Isolate Tv was iso-
lated as a root endophyte, while isolate T4 colonized the
rhizosphere. Isolates T2 and T4 were T. asperellum, T15 and T16
were T. longibrachiatum, and T1 and Tv were T. virens (Fig. 1).
DNA sequences of the amplified TEF1 were uploaded to GenBank
(JX15248–JX15260).
3.2. Trichoderma inhibits growth of Phytophthora in pre-colonized
plate assay
Only Phytophthora was isolated from Phytophthora only control
plates, confirming that there was no contamination in the experi-
ment (Tables 1 and 2). Trichoderma isolates T1, T2, T4, and Tv com-
pletely inhibited or killed P. tropicalis (Table 1) and P. palmivora
(Table 2) mycelium on precolonized V8 plates. T16 was only par-
tially antagonistic to the Phytophthora as P. tropicalis mycelium
was recovered from 61% of the plugs (Table 1) and P. palmivora
was isolated from 88% of plugs (Table 2). T15 lacked the ability
to effectively colonize P. tropicalis mycelium, as it was only isolated
from 50% of plugs (Table 1) and had limited efficacy against P. pal-
mivora (Table 2). T15 and T16 were slow growing isolates, lacking
the ability to completely colonize P. palmivora (Table 2).
3.3. Trichoderma isolates were only antagonistic to P. tropicalis in the
dual plate assay
All six Trichoderma isolates reduced P. tropicalis growth rate in
the dual plate assay (Fig. 2A). T1 suppressed P. tropicalis growth
T. virens T1 JX315249
T. virens Tv JX315248
T. virens EU280065
T. virens EU280060
T. virens FJ463366
T. virens GU591800
T. virens AY750894
T. harzianum AF348107
T. harzianum FJ463320
T. stromaticum AY937434
T. stromaticum HQ342166
T. asperellum EU338333
T. asperellum EU856323
T. asperellum T2 JX315260
T. asperellum T4 JX315259
T. asperelloides GU198240
T. asperellum AB568374
T. asperellum EU279957
T. asperelloides DQ381958
T gracile JN175598
T. longibrachiatum EU401597
T. longibrachiatum AY865640
T. longibrachiatum EU280033
T. longibrachiatum JN175567
T. longibrachiatum JN175564
T. longibrachiatum T15 JX315252
T. longibrachiatum T16 JX315257
Sphaerostilbella aureonitens FJ467644
0.10
0.01
0.02
0.01
0.01
0.00
0.27
0.10
0.03
0.00
0.01
0.17
0.07
0.01
0.01
0.07
0.01
0.04
0.05
0.04
0.02
0.06
0.00
0.05
0.05
Fig. 1. Phylogenetic tree inferred by Parsimony analysis of the tef1 gene sequences from Trichoderma isolates collected from the leaves and roots of cacao from trees in Aceh,
Indonesia.
36 R. Sriwati et al. / Biological Control 89 (2015) 33–41
more than all other isolates (Fig. 2A) at 32%. Although not signifi-
cantly different from each other, T2, T4, T15, T16, and Tv reduced
the growth of P. tropicalis by 21.3%, 17.3%, 20.0%, 18.7%, and
22.7%, respectively. None of the Trichoderma spp. inhibited the
growth rate of P. palmivora or reduced the overall diameter of
the P. palmivora colony throughout the experiment (data not
presented).
3.4. T. virens isolate Tv was antagonistic to Phytophthora in
mycoparasitism glass slide assays
Trichoderma T1, T2, T4, T15, and T16 did not coil around or
directly penetrate Phytophthora hyphae. In the absence of
Trichoderma (Fig. 3A) and in the presence of T2, T4, T15 (Fig. 3C),
and T16 P. palmivora readily produced normal sporangia. P. tropi-
calis rarely produced zoospores under the culture conditions used.
In the presence of T1, P. palmivora often had encysted zoospores
which were germinated (Fig. 3B). Only Tv was observed coiling
around the P.tropicalis hyphae (Fig. 3D). Tv mycelium surrounded
P. palmivora sporangia (Fig. 3E) and sporangia appeared to be
abnormally shaped and ruptured (Fig. 3F).
3.5. Trichoderma culture filtrates were antagonistic to Phytophthora
spp.
MIN salt concentration was significant (p< 0.0001) in affecting
the radial growth of P. tropicalis and P. palmivora. Despite this con-
centration effect, there was no significant interaction between fil-
trate concentration and isolate for either species. Neither P.
tropicalis (Table 3) nor P. palmivora (Table 4) were completely
inhibited by the culture filtrates of the Trichoderma isolates.
Culture filtrates from all six Trichoderma isolates reduced the
growth rate of P. tropicalis (Table 3). All isolates inhibited the
growth of P. palmivora except T1 (Table 4). Tv caused the greatest
inhibition of both Phytophthora spp. (Tables 3 and 4).
The Trichoderma isolates also affected the growth habit/appear-
ance of P. palmivora mycelium in the antibiosis assay. The P. palmi-
vora mycelium growth habit on T1 and T2 extract was similar to
that on unamended control plants (Fig. 4). P. palmivora on T4,
T15 and T16 extract amended media grew as a tuft from the plug
with limited extension into the media, while those of Tv had virtu-
ally no growth on the plug and very sparse limited extension into
the media (Fig. 4).
3.6. Trichoderma reduced black pod lesion in detached pod assay
Of all isolates tested, only Tv reduced P. palmivora lesion expan-
sion on detached cacao pods (Table 5). T. virens did not slow initial
lesion expansion, at one to two days after inoculation, but slowed
further lesion expansion. Overall, T. virens isolate Tv reduced final
lesion size by 77% after four days and reduced overall disease pro-
gression, measured by AUDPC, by 67% (Table 5).
3.7. T. virens isolate Tv reduced Phytophthora seedling blight
The concentration of T. virens isolate Tv applied to cacao seed-
lings affected the efficacy of biological control. Of the three spore
suspensions applied to cacao seedlings, only 1 10
8
conidia/ml
of Tv was capable of reducing the incidence of P. palmivora lesions
at 14 days after inoculation when compared to untreated control
plants (data not presented). While 1 10
4
CFU/ml did not
Table 1
Trichoderma isolates and Phytophthora tropicalis recovered from Phytophthora colo-
nized plates in which a strip of Trichoderma was overlaid at the edge and incubated for
one week. Plugs were removed at 0, 2, and 4 cm from the challenge point, plated and
onto MIN plates to determine whether the Trichoderma isolates were mycoparasites.
Isolates are T. asperellum T2 and T4, T. longibrachiatum T15 and T16 and T. virens T1
and Tv. Percentage below are the mean percentage of plates from eight replicate
plates from two combined experiments.
Treatments Percentage of spots microbe were
recovered
Trichoderma P.tropicalis Trichoderma (%) P.tropicalis (%)
T1 100 0
T2 100 0
T4 100 0
T15 100 0
T16 100 0
Tv 100 0
Control + 0 100
T1 + 100 0
T2 + 100 0
T4 + 100 0
T15 + 50 100
T16 + 100 60.8
Tv + 100 0
Table 2
Trichoderma isolates and Phytophthora palmivora recovered from Phytophthora
colonized plates in which a strip of Trichoderma was overlaid at the edge and
incubated for one week. Plugs were removed at 1, 4, and 8 cm from the challenge
point, plated and onto MIN plates to determine whether the Trichoderma isolates
were mycoparasites. Isolates are T. asperellum T2 and T4, T. longibrachiatum T15 and
T16 and T. virens T1 and Tv. Percentage below are the mean percentage of plates from
eight replicate plates from two combined experiments.
Treatments Percentage of spots microbe were
recovered
Trichoderma P.palmivora Trichoderma (%) P.palmivora (%)
T1 100 0
T2 100 0
T4 100 0
T15 100 0
T16 100 0
Tv 100 0
Control + 0 100
T1 + 100 0
T2 + 100 0
T4 + 100 0
T15 + 79 67
T16 + 83 88
Tv + 100 0
P. tropicalis growth rate (cm/day)
Control T1 T2 T4 T15 T16 Tv
Treatments
Fig. 2. Mean growth rate of Phytophthora tropicalis (A) and P. palmivora (B) in the
dual plate assay on CMDA to screen whether Trichoderma spp. were antagonistic to
Phytophthora. Bars extending from the mean indicate the standard error.
Differences in letters above the bar indicate statistically significant differences via
Tukey analysis.
R. Sriwati et al. / Biological Control 89 (2015) 33–41 37
significantly reduce the percentage of infected leaves, application
of 1 10
6
and 1 10
8
CFU/ml of T. virens isolate Tv reduced the
percentage of leaves infected by P. palmivora (Fig. 5).Increasing
concentrations of conidia, improved disease reduction as
110
8
CFU/ml reduced the number of infected leaves by 58.0%
and 1 10
6
CFU/ml only reduced infection by 36.6% compared to
untreated control (Fig. 5).
4. Discussion
While Trichoderma has long been studied as a soil and root col-
onizer, its use in aerial applications, in particular in tropic environ-
ments, is a recent area of study. The three Trichoderma species
isolated in Aceh in this experiment, T. virens,T. asperellum, and T.
longibrachiatum, have all been previously identified in Indonesia
(Kubicek et al., 2011). However, their role in the cacao agroecosys-
tem is not well studied. Despite Indonesian and South American
cacao plantations facing different pests, these species have also
been previously identified as mycoparasites and endophytes of
Theobroma spp. in South America, the center of origin for the cacao.
T.asperellum was isolated as an endophyte of cacao in Brazil along
with T.longibrachiatum (Hanada et al., 2009). T. longibrachiatum is
an epiphytic mycoparasite in cacao fields in Peru (Krauss and
Soberanis, 2002). T. longibrachiatum is a cosmopolitan species that
is predominantly tropical (Samuels et al., 2012). In a study on the
endophytes in Central Sulawasi, Indonesia, there was a decreased
diversity of fungal endophytes inhabiting cacao trees in compar-
ison to those in South America (Schmidt et al., 2010).
Trichoderma has been well studied as a biological control agent
(Harman et al., 2004), with an increasing amount of work studying
Trichoderma to manage cacao diseases (Bae et al., 2009; Bailey
et al., 2006; de Souza et al., 2006; Deberdt et al., 2008; Hanada
et al., 2009; Krauss et al., 2010). All studied Indonesian
Trichoderma isolates were antagonistic to P. tropicalis in the dual
plate assay, but at varying levels, while none were inhibitory to
P. palmivora.T. longibrachiatum isolates T15 and T16 had limited
to no activity against either Phytophthora spp. in the precoloniza-
tion assays. It should be noted that both T15 and T16 grew more
slowly than the other isolates. The lack of inhibition by T15 and
T16 in the precolonized plate assay may have been due to their
reduced growth rate, as fast growing isolates T1, T2, T4, and Tv
eliminating both P. tropicalis and P. palmivora.
The isolates of Trichoderma studied varied greatly in their abil-
ities to produce compounds inhibitory to the Phytophthora spp.
Filtrates from isolates T2, T4, T15, T16, and Tv inhibited the growth
of P. tropicalis and P. palmivora, with Tv filtrates having the most
Fig. 3. Interaction zone between endophytic Trichoderma virens isolate Tv and Phytophthora tropicalis and P. palmivora on a thin layer of water agar on a glass slide. Slides were
incubated for seven days and observed daily for presence of the interaction zone, coiling of Trichoderma around Phytophthora and malformation in hyphal growth. Slides were
stained with lactophenol cotton blue to aid in visualization. (A) P. palmivora sporangia in the absence of Trichoderma. (B) P. palmivora sporangia lacking zoospores and
germinating in the presence of T1. (C) T15 comingling with P. palmivora without causing a noticeable reaction. (D) T. virens isolate Tv hyphae coiling around P.tropicalis. (E)
Ruptured misshapen round P. palmivora sporangia surrounded by T. virens isolate Tv mycelia. (F) Ruptured P. palmivora sporangia in the presence of T. virens isolate Tv. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 3
Mean growth rate (mm/day) of P.tropicalis in the antibiosis assay to screen whether
heat treated culture filtrates of the Trichoderma spp. were capable of inhibiting the
growth of Phytophthora when added to plates at a 1:1 into 1and 2MIN media.
MIN salt concentration was significant.
Isolate Mean P. tropicalis growth (mm/day)
1MIN 2MIN
Control 5.0A
a
5.1A
T1 2.8E 2.7C
T2 4.0C 3.9B
T4 4.2BC 3.7B
T15 3.5D 3.6B
T16 4.4B 4.1B
TV 2.3F 1.9D
a
Differing letter within a column indicate significant difference determined by
Tukey analysis at p60.05.
Table 4
Mean growth rate of P.palmivora (mm/day) in the antibiosis assay to screen whether
heat treated culture filtrates of the Trichoderma spp. were capable of inhibiting the
growth of Phytophthora when added to plates at a 1:1 into 1and 2MIN media.
MIN salt concentration was significant.
Isolate Mean P. palmivora growth (mm/day)
1MIN 2MIN
Control 5.5A
a
6.5A
T1 4.7AB 6.0A
T2 3.9BC 5.0B
T4 3.9BC 3.8C
T15 3.0CD 3.0DE
T16 3.2C 3.3CD
TV 2.2D 2.6E
a
Differing letter within a column indicate significant difference determined by
Tukey analysis at p60.05.
38 R. Sriwati et al. / Biological Control 89 (2015) 33–41
activity in inhibiting the growth of both Phytophthora spp.
Trichoderma species produce many antimicrobial compounds
(Howell, 2003, 2006). Cell free extracts of T. virens have previously
been shown to inhibit Phytophthora such as the solanaceous patho-
gen Phytophthora erythroseptica (Etebarian et al., 2000), forest
pathogen Phytophthora ramorum (Becker et al., 2011), soybean
pathogen Phytophthora sojae (Ayoubi et al., 2012). It should also
be noted that gliotoxin produced by some strains of T. virens has
the potential to inhibit both Pythium and Phytophthora spp.
(Howell, 2006).
Tv was the only isolate observed to coil around P. tropicalis
hyphae and to disrupt P. palmivora sporangia. The P. palmivora
mycelium was very thin on water agar slides, making it difficult
to observe the interaction between P. palmivora mycelia and
Trichoderma mycelia. At the same time, P. palmivora produced a
large numbers of sporangia on the water agar slide, not seen in P.
tropicalis.Trichoderma spp. are known to parasitize the hyphae of
many fungi and oomycete species (Almeida et al., 2007; Lu et al.,
2004; Rocha-Ramírez et al., 2002). In doing so, Trichoderma can
penetrate and destroy hyphae and resting structures, which
reduces the infective potential of the pathogen (Almeida et al.,
2007). In interactions with Phytophthora cinnamomi, several
Trichoderma species were seen to coil around the P. cinnamomi
hyphae, but only one Trichoderma isolate penetrated the hyphae
(Aryatha and Guest, 2006). The coiling that was observed in these
experiments was limited, but the disruption of the sporangia was
extensive, possibly due to antimicrobial compounds produced by
Tv.
On detached cacao pods sprayed with the Trichoderma isolates,
T. virens isolate Tv reduced the lesion diameter compared to all
other treatments. Trichoderma has previously been shown to
reduce black pod of cacao (Hanada et al., 2008; Tondje et al.,
2007). T. virens GL-21, commercially sold as SoilGuard, did not
reduce black pod caused by P. palmivora or increase pod yield in
field trials in Peru (Krauss and Soberanis, 2001). To best utilize
Trichoderma to manage black pod rot, a better understanding of
Trichoderma growth on pod surfaces is needed as increased knowl-
edge of colonization location and longevity is needed to optimize
use in the field. Additionally, work on formulations can increase
the likelihood of germination of conidia and survival of subsequent
Trichoderma hyphae.
T. virens isolate Tv was not antagonistic to P. palmivora in the
dual plate assay, despite its reducing P. palmivora lesions on the
detached pod and in the seedlings assays. These results are an
example in which completing only a dual plate assay to screen
Fig. 4. P. palmivora hyphae grown on MIN broth containing culture filtrates from
each of the Trichoderma isolates. Filtrates were incubated at 90 °C for two hours to
inactivate enzymes. MIN plates were incubated at 25 °C and visualized after one
week of growth.
Table 5
Mean lesion diameter of and AUDPC black pod rot spots on cacao pods inoculated
with 1 10
6
conidia/ml and challenged with P. palmivora one day later. Treatments
are water control and isolates T. asperellum T2 and T4, T. longibrachiatum T15 and T16,
and T. virens T1 and Tv. Data below are eight replicate pods from two combined
experiments.
Trichoderma treatments Days after inoculation of P. palmivora
a
Day 1 Day 2 Day 3 Day 4 AUDPC
Control 1.1 A 1.7 ABC 3.2 A 6.0 A 9.0 A
T1 1.2 A 1.3 ABC 2.9 A 5.7 A 8.2 A
T2 1.4 A 1.4 A 3.2 A 6.1 A 9.0 A
T4 0.7 A 1.6 ABC 2.7 A 5.4 A 7.7 A
T15 0.9 A 1.8 ABC 2.9 A 5.4 A 8.3 A
T16 0.5 A 1.9 BC 2.2 A 5.1 A 7.1 A
Tv 0.3 A 0.7 C 1.1 B 1.4 B 2.8 B
a
Differing letter within a column indicate significant difference determined by
Tukey analysis at p60.05.
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
Disease Incidence (% of leaves with symptoms)
Concentrations of T. v ir en s isolate Tv (conidia/ml)
A
A
B
B
1x1041x1061x108
Control
Fig. 5. Mean percentage of cacao leaves infected by P. palmivora after application of
T. virens isolate Tv at 1 10
4
,110
6
, and 1 10
8
conidia/ml to 15 days old cacao
seedlings. Seven days after application of T. virens, the seedlings were inoculated
with 1 10
6
sporangia/ml of P. palmivora and observed for overall disease
incidence, the number of infected leaves, and the total number of leaves two
weeks after application. Bars extending from the mean indicate the standard error.
Differences in letter above the bar indicate statistically significant differences via
Tukey analysis.
R. Sriwati et al. / Biological Control 89 (2015) 33–41 39
isolates would have eliminated the best isolate. Testing isolates via
multiple screening assays, as has been previously done (Bailey
et al., 2006; Evans et al., 2003; Melnick et al., 2011) allowed for a
better selection of isolates and a better understanding of the mode
of action of the potential biological control agent.
The additional studies on Phytophthora seedling blight further
showed the potential of Tv in managing that phase of the cacao dis-
ease in Indonesia. Phytophthora seedling blight causes significant
losses in areas where cacao is produced (Bowers et al., 2001).
Although an issue in nurseries, the use of seedling assays as a
screening method for cacao biological control agents is not well
studied. T. virens isolate Tv was capable of reducing Phytophthora
seedling blight when applied at high concentrations
(1 10
8
conidia/ml).
While we cannot fully determine the mode of action in these
studies, our laboratory work supports the ability to T. virens isolate
Tv to be antagonistic in petri dishes, produce antibiotic substances
that inhibit growth, and act as a mycoparasite. T. virens isolate Tv is
a mycoparasite of Phytophthora, antagonizes P. tropcalis in the dual
plate assay, and produces metabolites antagonistic to both P.trop-
icalis and P.palmivora, suggesting that multiple modes of action
contribute to its ability to limit black pod rot and seedling blight
of cacao. While T. virens isolate Tv shows potential as a biological
control agent for P. palmivora, future work should focus on improv-
ing survival of conidia in the environment through formulation.
We plan to develop an invert oil emulsion (Womack et al., 1996)
utilizing oil that is readily found in the region as a formulation
for the product. We also plan to conduct experiments with larger
seedlings as well as scaling up to field-level testing to determine
whether T. virens isolate Tv will act as a biological control agents
of black pod rot in the field with the ultimate goal of improving
the production in Aceh Indonesia. (Abo-Hamed et al., 1981).
Acknowledgments
USDA is an equal opportunity provider and employer. The
World Cocoa Foundation and the WCF-Aceh Cacao Fellowship
Program under SwissContact’s PEKA Project supported Dr.
Swarti’s travel and living expenses while at the USDA-ARS-SPCL.
Fieldwork was supported by a Grant received from the
SwissContact’s PEKA Project.
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