Biological Control 141 (2020) 104145

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

Trichoderma asperellum biocontrol activity and induction of systemic T

defenses against Sclerotium cepivorum in onion plants under tropical climate
conditions
William Rivera-Méndeza,b, Miguel Obregónc, María E. Morán-Diezb, Rosa Hermosab,
⁎
Enrique Monteb,
a
Costa Rica Institute of Technology (ITCR), Calle 15, Avenida 14, Cartago, Costa Rica
b
Spanish-Portuguese Institute for Agricultural Research (CIALE), Department of Microbiology and Genetics, University of Salamanca, C) Duero 12, 37185 Salamanca,
Spain
c
Laboratorios Dr. Obregón, 350m Oeste del Hospital nuevo de Heredia, Heredia, Costa Rica

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: The biocontrol potential of three native Costa Rican Trichoderma asperellum strains has been evaluated against
White rot the necrotrophic ascomycete Sclerotium cepivorum, the causal agent of onion (Allium cepa L.) white rot. In Costa
Antagonism Rica, where climatic conditions enhance the development of this pathogen, white rot reduces onion yields up to
Systemic resistance 50% of total harvest. In our study, the three T. asperellum strains tested in in vitro assays showed their capacity to
Onion bulb yield
antagonize S. cepivorum, with BCC1 displaying percentages of colony growth inhibition of the pathogen of 81
and 90% in dual culture and cellophane membrane assays, respectively. In addition, this Trichoderma strain was
able to reduce a 74% the plant mortality compared to untreated plants under greenhouse conditions. In field
trials, carried out in two consecutive harvest years and in two different tropical locations, the two tested dosages
of T. asperellum BCC1 reduced the incidence of white rot in a 3.41% for the lowest dose and 3.61% for the highest
dose when compared to onion plants treated with chemical fungicides. Additionally, a significantly increase of
20.4% in onion bulb yield was recorded for the highest dose of BCC1. The potential of T. asperellum BCC1 to
induce systemic defenses in onion plants against S. cepivorum was evaluated for four onion defense marker genes
in a 21-day time course study by quantitative real-time PCR and using onion plants grown under greenhouse

⁎
Corresponding author at: Spanish-Portuguese Institute for Agricultural Research (CIALE), Department of Microbiology and Genetics, University of Salamanca, C)
Duero 12, 37185 Salamanca, Spain.
E-mail addresses: [email protected] (W. Rivera-Méndez), [email protected] (M. Obregón), [email protected] (M.E. Morán-Diez),
[email protected] (R. Hermosa), [email protected] (E. Monte).

https://doi.org/10.1016/j.biocontrol.2019.104145
Received 1 July 2019; Received in revised form 28 October 2019; Accepted 7 November 2019
Available online 08 November 2019
1049-9644/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
W. Rivera-Méndez, et al. Biological Control 141 (2020) 104145

conditions. The expression profile of AcPR1 and AcPAL1, characterized by being undulating, indicates an initial
activation of salicylic acid-dependent defense pathways (1 and 7 days) by T. asperellum BCC1 when either ap-
plied alone or in combination with the pathogen, while the up-regulation of AcEIN3 observed in those same
treatments at day 21 revealed the activation of ethylene-dependent defense pathways by this Trichoderma strain.
T. asperellum BCC1 exerts efficient biocontrol against S. cepivorum and activates onion systemic defenses against
this pathogen under greenhouse conditions, while it reduces onion white rot incidence and increases crop yield
in field trials performed under Costa Rica’s tropical climate conditions.

1. Introduction mediated by salicylic acid (SA) (Martínez-Medina et al., 2013; Morán-

Diez et al., 2009; Salas-Marina et al., 2011; Tucci et al., 2011). In early
Onion (Allium cepa L.) is a profitable vegetable crop grown for the works, Metcalf et al. (2004) tested Trichoderma koningii as an antagonist
appreciated attributes of its bulbs associated to flavor and health ben- to protect onion roots, and Clarkson et al. (2004) working with Tri-
efits. Onion is also considered among the most consumed vegetables choderma viride and Trichoderma pseudokoningii, achieved 80% of
worldwide, being exceed only by tomatoes (Havey, 2018). Its global sclerotia parasitization. More recently Hussain et al. (2017) reported
area harvested reached more than 5 million ha and a production of near the use of T. viride, Trichoderma hamatum and Trichoderma harzianum to
100 million tons in 2017 (FAO, 2017), with a market estimated in 18 control white rot in onion plants with an efficiency that was close to
billion U.S. dollars in 2014 (Havey, 2018). In terms of cultivable land, 75% under greenhouse conditions. However, studies focusing on the
Costa Rica’s tropical climate allows for the successfully cultivation of mechanisms underlying the induction of onion defense responses
different onion varieties. Most of the production in this country is mediated by Trichoderma asperellum against S. cepivorum in onion plants
concentrated in the northern regions of the province of Cartago at al- are not available. Furthermore, there are not evidences of the effect of
titudes higher than 1700 m in a tropical mountain ecosystem, and onion T. asperellum on the control of onion white rot and crop yield in field
is considered a valuable commodity for the local markets with a total trials in tropical agro-ecosystems.
production of 33,000 tons per year (Serrano and Morales, 2017). The aim of this study was to evaluate the biocontrol potential of T.
The soil-borne ascomycete fungus Sclerotium cepivorum Berk (tele- asperellum strains against S. cepivorum in onion plants. To achieve this
omorph: Stromatinia cepivora) is an economically important necrotroph goal, in vitro antagonism assays served to select the most promising T.
pathogen that causes the white rot disease of several Allium species, this asperellum strain, which was further tested in in vivo greenhouse assays,
being a limiting factor to the onion production worldwide (Hanci, in terms of plant fitness, biocontrol of S. cepivorum and activation of
2018; Havey, 2018; Hovius and McDonald, 2002). S. cepivorum has defenses against this pathogen, and in field trials under Costa Rica’s
white mycelium without conidial development, but in absence of a host tropical climate conditions, in terms of biocontrol efficiency and crop
plant it persists as a survival structure, called sclerotia, which can yield. To our knowledge, neither biocontrol of white rot in onion bulb
survive in the soil for up to 20 years (Coley-Smith et al., 1990). These data nor studies with onion-defense marker genes have been reported
compact masses of black-hardened mycelia, with a spherical shape and under this environmental scenery what makes of this research study a
range in size from 200 to 500 μm in diameter, are the primary source of significant contribution to this field.
infection of the pathogen (Elsherbiny et al., 2015). Allium root exudates
play an active role in the release of volatile organic compounds which 2. Materials and methods
stimulate germination of the sclerotia (Coley-Smith and King, 1969).
Once the germination occurs hyphae grow through the soil until en- 2.1. Fungi and onion seeds
countering the onion plant, penetrating the roots and destroying their
radicular system. In Costa Rica, especially in the province of Cartago, Strains BCC1, BCF2 and BCF7 of Trichoderma asperellum, isolated
with soils rich in organic matter and very stable environmental condi- from onion roots in production fields from the district of Llano Grande
tions throughout the year, the white rot is the most relevant disease and (Cartago, Costa Rica), were used as the antagonistic fungi. Sclerotium
has negative effects on crop yield with losses reaching the 50% of ex- cepivorum SCM strain, previously characterized as a highly virulent
pected production (Aguilar-Ulloa et al., 2016). pathogen to onion plants, was used as target in our experiments. Fungal
Control of white rot disease is complicated as it has to face a number strains were routinely grown on potato dextrose agar (PDA) (Difco,
of biological and environmental challenges. The use of plant varieties Franklin Lakes, NJ, USA) on Petri dishes for 8–14 days at 22–25 °C in
with genetic resistance to the pathogen would be the preferred chosen the dark. T. asperellum strains were preserved in 30% glycerol conidia
method of controlling this disease, though these onion germplasms are suspension at −80 °C, and S. sclerotiorum SCM strain was stored as PDA
not widely deployed in commercial cultivars (Havey, 2018). Ad- plugs suspended in sterile water at 4 °C. Fungi are deposited in the
ditionally, the use of agrochemicals has been poorly efficient, due to the culture collection at the Biocontrol Laboratory in the Costa Rica
accumulated resistance by the broad application of a limited number of Institute of Technology (ITCR) of Cartago.
registered molecules, which has favored the use of other methods like Onion seeds (Allium cepa “Alvara”) (Bejo Eurosemillas, Costa Rica)
the introduction of biological control agents, especially Trichoderma were surface-sterilized by vigorous sequential shaking in 70% ethanol
strains (McLean et al., 2012). and 2% sodium hypochlorite solutions for 10 min each, then washed
Biological control mechanisms displayed by Trichoderma species thoroughly four times in sterile distilled water, and air-dried on a sterile
have been studied for decades (Harman et al., 2004; Lorito et al., 2010). gauze sheet. This onion variety was used throughout this study.
These non-pathogenic soil-borne microorganisms have the ability to
antagonize and mycoparasitize fungal pathogens, to colonize the plant
2.2. Fungal growth inhibition assays
radicular system and to induce plant defense responses (Hermosa et al.,
2012). Phytohormone-mediated defenses are known as systemic ac-
Confrontation assays (dual cultures) between T. asperellum strains
quired resistance (SAR) and induced systemic resistance (ISR) (Pieterse
BCC1, BCF2 and BCF7, and S. cepivorum SCM strain were performed as
et al., 2009). As plant beneficial fungi, Trichoderma spp. are known by
previously described (Rubio et al., 2009) with some modifications.
their capability to trigger ISR responses based on jasmonates (JA) and
Briefly, a 5 mm-diameter agar plug colonized by S. cepivorum was
ethylene (ET)-dependent pathways (Hermosa et al., 2012). It has been
grown on Petri dish at 23 °C in the dark for 3 days before placing a
also reported that some Trichoderma species trigger plant defenses
5 mm-diameter agar plug colonized by a T. asperellum strain at 2 cm

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W. Rivera-Méndez, et al. Biological Control 141 (2020) 104145

from the border on the opposite side of the plate. Cultures of the SCM plants was collected from each treatment and each considered time (1,
strain growing alone were used as controls. After 12 days of dual cul- 2, 7, and 21 days), and immediately frozen for further experiments of
ture, the mycelial growth diameter of the pathogen was measured and real-time quantitative PCR (RT-qPCR) and phenylalanine ammonia-
the percentage of fungal inhibition (FI) was calculated according to lyase (PAL) activity measurements. The greenhouse assay was repeated
Royce and Ries (1978). FI (%) = RGI × 100; RGI = (C-T)/C; where T is twice.
the average diameter of mycelial growth in the presence of a T. asper-
ellum strain, and C is the average diameter of the mycelial growth in the 2.3.3. Biocontrol assays under field conditions
control plates. The assays were performed in triplicate. Experimental plots were established in two farms (referred to here
Membrane antifungal assays on cellophane sheets were carried out as “Tierra Blanca“ and ”Llano Grande“) in the province of Cartago in
on PDA medium and in triplicate as previously described (Rubio et al., Costa Rica, under the climatic conditions of the tropical mountain.
2009). The diameters of the fungal colonies were measured after 5 days “Tierra Blanca” soil was characterized as silt loam (pH 5.9) while
of incubation at 23 °C in the dark. Results were expressed as the growth “Llano Grande” was clay loam with a pH of 5.7. The experiments were
inhibition percentage of S. cepivorum SCM strain by each T. asperellum carried out in the rainy season of years 2017 and 2018. Onion plants
strain tested, BCC1, BCF2 and BCF7, with respect to the mean colony mortality caused by S. cepivorum and total yield were evaluated under
diameters of the SCM strain grown alone. these conditions. The initial inoculum level of S. cepivorum was estab-
lished according to the method of Papavizas (1972). Briefly, 50 g of soil
2.3. In-planta assays were mixed with 200 mL of distilled water in a blender at 2000 rpm and
sieved through 20-mesh and 80-mesh sieves. The residue on the 80-
2.3.1. Plant growth promotion by T. asperellum mesh sieve was washed with running tap water for 15 min and later
An in vitro assay was carried out to analyze the effect of strains dried in an oven for 1 h at 50 °C. Sclerotia were observed under the light
BCC1, BCF2 and BCF7 of T. asperellum on onion seedlings growth. microscopy and their number was expressed as sclerotia per g of soil.
Onion seeds, previously surface-sterilized, were grown in Murashige Fifty-days-old onion seedlings, grown in a mixture of peat:soil (3:1)
and Skoog (MS) medium (Duchefa Biochemie, Haarlem, The under greenhouse conditions as described above, were used in these
Netherlands), supplemented with 1% sucrose and 0.8% agar (pH 5.7), field assays. Three treatments were considered: T1, control; T2 and T3,
for 3 days. Each biological replicate included one plate with four seeds T. asperellum used at two different concentrations. For control treatment
per tested strain. Conidia from 7-day-old PDA plates were harvested by (T1), onion plants were sprayed with a mixture (1:1 ratio) of the fun-
adding 5 mL of water to the plates and scraping the culture with a gicides carboxin and captan (Vitamax 400®, Arysta Lifesciences, Salt-
rubber spatula. These suspensions were filtered through a double layer illo, Mexico) in the field at the initial stage of the experiment, and with
of cheesecloth to separate large mycelial fragments from conidia. the fungicide tebuconazole (Folicur 25 EC®, Bayer CropScience, San
Conidia concentrations were calculated using a counting chamber and José, Costa Rica) after 30, 45, 60, and 75 days of planting. Carboxin-
suspensions were used to inoculate the MS medium. An inoculum captan was applied at a rate of 0.25% (w/v) of the product per ha of
containing 1 × 106 conidia of each Trichoderma strain was placed at the soil, and tebuconazole was used as 0.12% (v/v) of the product per ha of
opposite side of the plates containing 3-day-old onion seedlings. Plates soil. Treatment T2, onion plants treated by root immersion in 4 L of a
were cultured in a growth chamber under conditions of 40% humidity, conidial suspension (1 × 106 conidia/mL) of T. asperellum BCC1 for
24 °C, and a 16-h light/8-h dark photoperiod. Plates containing only 5 min, then planted in the field soil, and later an additional sprayed of
onion seedlings, without Trichoderma conidia, and grown under similar 4 mL of 1 × 106 BCC1 conidia/mL were applied per plant at 30, 45, 60,
conditions were used as control. Experiments were performed in tri- and 75 days after planting; for T3, onion plants were treated similarly to
plicate, and measurements of onion seedlings roots and aerial sections those of T2 treatment but instead a concentration of 1 × 107 conidia/
were recorded 5 days after Trichoderma inoculation. mL of BCC1 was used. Each experimental unit was a 1.1 × 1.1 m2 with
a density of 100 plants/m2. For each treatment, 10 repetitions were
2.3.2. Biocontrol assays under greenhouse conditions made with 100 plants in each repetition. In addition to the treatments
Biocontrol tests were carried out with T. asperellum BCC1 and S. applied, each unit received the same general care with applications of
cepivorum SCM in onion plants under greenhouse conditions. Two- granular NPK fertilizer of 10-30-10 formula at 30 days, of 20-20-20 at
hundred onion plants were grown in a mixture of peat:soil (3:1) for 60 days, and of 12-11-17 at 90 days. In addition, a solution of 400 mL/
50 days before being transplanted and used in this assay. The plants m3 of calcium nitrate (20 g of active compound per kg of commercial
were planted individually in pots of 0.7 L capacity using a sterile product) (AugeCalcio 18®, AgroPro Centroamérica, San José, Costa
mixture of peat:soil (3:1) supplemented with smart-release fertilizer Rica) was sprayed on the leaves, and 4 mL of an acaricide (Akaramic 1.8
(Osmocote®, dosage 4 kg/m3 of substrate) (Agrotico, Cartago, Costa EC®, based on abamectin B1a and B1b) (0.25% v/v) (Rotam CropS-
Rica). Four different treatments (T1 to T4) were considered, using 50 ciences, Hong Kong, China) were applied per plant 70 days after
onion plants per treatment, in a completely randomized test: T1, control planting. Total mortality and final yield were recorded at the last stage
(untreated); T2, plants treated with T. asperellum BCC1; T3, plants in- of the experiment, 5 months after initial planting.
fested with S. cepivorum SCM; and T4, plants treated with both fungal
strains. For treatments T2 and T4, 10 mL of a conidial suspension of T. 2.4. Real-time quantitative PCR (RT-qPCR)
asperellum BCC1, with a concentration of 1 × 107 conidia/mL, was ap-
plied to the roots of 50-days-old onion plants. For treatments T3 and T4, Expression analyses were performed with onion plant leaves sam-
10 g of sand containing 100 S. cepivorum sclerotia/g were mixed with pled from the greenhouse assay described above. The central leaf of five
the sterile mixture peat:soil (3:1) per pot, and 57-days-old onion plants plants was separately collected for each treatment at 1, 2, 7 and 21 dpi
were transplanted. Thus, plants of T4 treatment were infested with the and considered as biological replicate. Total RNA was extracted using
pathogen one week after Trichoderma application, and similar infection the GenElute Universal Total RNA Purification Kit® (Sigma-Aldrich, St.
timing was followed for plants in T3, except no T. asperellum was ap- Louis, MO, USA) and then used to synthetize cDNA with the
plied to them before. The plants were maintained in a greenhouse at ReadyScript cDNA Synthesis Mix® (Sigma-Aldrich) following
23 ± 4 °C, and watered as needed. Fifteen days post-inoculation (dpi) manufacturés recommendations. The PCR reaction was performed on a
with the pathogen, root and aerial part lengths as well as their re- Light Cycler 480® (Roche Molecular Diagnostics, Pleasanton, CA, USA)
spective dry weights were recorded for 10 plants in each treatment. and using the Light Cycler 480 SYBR Green I Master® (Roche
Mortality was evaluated 21 dpi in 20 plants per treatment. For ex- Diagnostics GmbH, Mannheim, Germany). Reaction mixtures and am-
pression levels of defense marker genes, the central leaf of five onion plification conditions were carried out as previously described

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W. Rivera-Méndez, et al. Biological Control 141 (2020) 104145

(Montero-Barrientos et al., 2011). The reaction was carried out in a Trichoderma were significantly shorter than those of the control treat-
total volume of 10 μL and three technical replicates for each tested ment, but there were not differences among the strains. Regarding the
condition. The Ct values were normalized with the values of the onion stem length, there were no differences between treatments, including
actin gene (AcACT) and the relative gene expression was calculated the control condition.
using the 2−ΔΔCT method (Schmittgen and Livak, 2008). Marker genes
of SA-, JA-, and ET-dependent defense pathways: AcPR1 and AcPAL1, 3.3. Biocontrol of onion white rot by T. asperellum BCC1 under greenhouse
AcLOX1, and AcEIN3 respectively were analyzed. The sequence of pri- conditions
mers used in this study and the GenBank access numbers of each gene
are listed in the Supplementary Material Table S1. The availability of The capacity of T. asperellum BCC1 to control onion white rot was
any genomic database for Allium is limited to the Onion Genomic Da- evaluated in 50-days-old onion plants. The root length and dry weight,
tabase (Shukla et al., 2016) and was used as a source of references for stem length, canopy dry weight and the mortality were evaluated in a
these genes. The Primer3Plus® software (Free Software Foundation Inc., greenhouse assay (Table 3). The analysis of root and stem growth
Boston, MA, USA) and the OligoAnalyzer 3.1 (Integrated DNA Tech- parameters, evaluated 15 dpi with the pathogen, revealed that onion
nologies, Skokie, IL, USA) were used to design and test in silico the plants infested with S. cepivorum alone displayed the lowest values
sequences. differing significantly from the other three treatments, while those
corresponding to the control and T. asperellum + S. cepivorum treat-
2.5. Determination of L-phenylalanine ammonia-lyase (PAL) activity ments showed the highest for the four growing traits measured. Onion
plants treated solely with BCC1 showed values of stem and root lengths
The central leaf from five plants of the greenhouse assay per treat- smaller than those of the control, though no significant differences were
ment and sampling time was processed as described above and used to observed for the corresponding dry weights. Mortality percentages were
determine the PAL activity. The plant material was homogenized in evaluated on 20 onion plants for each treatment 21 dpi with S. cepi-
3 mL of 0.1 M trisodium borate buffer (pH 8.5) containing 1.4 mM 2- vorum. As expected, control and T. asperellum treatments presented
mercaptoethanol and 0.1 g of insoluble polyvinylpyrrolidone. The ex- 100% healthy plants indicating that this Trichoderma strain did not
tract was filtered through cheesecloth and centrifugation was carried cause damage to any plant. Moreover, onion plants treated with BCC1
out at full speed for 15 min. The determination of PAL activity was showed a positive effect on the onion protection against S. cepivorum as
analyzed as the rate of conversion of L-phenylalanine into trans-cin- the ratio of mortality observed in onion plants treated with both fungal
namic acid at 270 nm in a spectrophotometer. A 200-μL aliquot of the strains was 14% compared to the 88% of mortality recorded in plants
sample was added to 400 μL of borate buffer and 200 μL of 40 mM L- solely infected with S. cepivorum.
phenylalanine, and the reaction mixture was incubated at 37 °C for
15 min. Reaction was stopped with an equal volume of 10% (w/v) of 3.4. Biocontrol of onion white rot by T. asperellum BCC1 under field
trichloroacetic acid (TCA), and samples were centrifuged at full speed conditions
for 15 min. The supernatants were used to measure the absorbance at
270 nm, and trans-cinnamic acid was calculated using a standard curve The initial inoculum determined for “Tierra Blanca” farmland was
according to Lee et al. (2015). Results are presented as μmol/min (U) 0.020 sclerotia/g of soil and for “Llano Grande” farmland it was 0.025
per g of fresh tissue. sclerotia/g of soil. Based on these values both soils were considered
homogeneous and suitable for trials.
2.6. Statistical analyses The field tests showed the effects of two doses of T. asperellum BCC1
on onion white rot biocontrol compared to those of agrochemicals
The data were analyzed using Minitab 18 Statistical Software commonly applied by farmers which were used as control (Tables 4 and
(Minitab, Inc., State College, PA, USA). Normality and homoscedasticity 5). For mortality analysis (Table 4), the GLM test determined that only
were evaluated. All data were analyzed with ANOVA followed by Fisher the type of treatment had a significant effect on this parameter. The
test (P < 0.05), with the exception of gene expression levels that were comparison test (Fisher method) determined that there were no dif-
analyzed by ANOVA and Tukey test (P < 0.05). Particularly, data re- ferences between the data from the two farms considered (P < 0.05),
corded from field assays were analyzed using a generalized linear but there were differences among the treatments. The control condition
model (GLM) with a significance of 5% and a Fisher comparison. presented the highest plant mortality (4.59 ± 0.39 plants/m2) which is
equivalent to 45,900 plants/ha or 4.59% of mortality. Onion plants
3. Results treated with the lowest concentration of spores of BCC1 (treatment T2)
showed the lowest mortality ratio (3.41%) while a higher dose in-
3.1. Antagonism of T. asperellum strains against S. cepivorum creased the plant mortality (3.61%). For yield analysis (Table 5), the
GLM test determined that both the year (P = 0.03) and the treatment
In both dual culture and cellophane membrane tests the three T. (P = 0.00) had a significant effect on onion yield (P < 0.05). The
asperellum strains reduced growth of S. cepivorum. Significant differ- lowest yield (2.45 ± 0.28/m2, equivalent to 24.5 ton/ha) was ob-
ences in percentages in FI of S. cepivorum were observed for the three tained with the control (treatment T1). On the other hand, the highest
different strains of T. asperellum evaluated (Table 1). In the dual culture dose of T. asperellum (treatment T3) produced the highest yield with
antagonism test, the strains BCC1 and BCF7 showed the highest FI
while strain BCF2 gave a significantly lower value. The antagonistic Table 1
capacity of Trichoderma strains was also analyzed in cellophane assays Percentage of growth inhibition of S. cepivorum SCM by three T. asperellum
that allowed to determine the effect of hydrolytic enzymes and meta- strains (BCC1, BCF2, and BCF7) in dual culture (12 days) and cellophane
bolites secreted to the culture medium. The strain BCC1 showed a membrane (5 days) assays. Values are means from three biological re-
significant highest FI percentage, compared to the other two strains, plicates ± standard deviations. Different letters indicate significant differences
(Fisher test, P < 0.05).
BCF2 and BCF7, which did not present significant differences.
T. asperellum Dual culture Cellophane membrane
3.2. Effect of T. asperellum strains on onion seedlings growth
BCC1 80.66 ± 2.76 a 89.86 ± 0.63 a
BCF2 69.39 ± 2.65b 83.70 ± 1.09b
The T. asperellum effect on the growth of 3-days-old onion seedlings BCF7 79.20 ± 1.09 a 81.52 ± 3.92b
was evaluated in in vitro tests (Table 2). Onion roots treated with

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Table 2 Table 4
Effect of three T. asperellum strains (BCC1, BCF2, and BCF7) on the growth of 3- Generalized linear model for oniońs mortality in field experiments.
day-old onion seedlings. Measurements of root and stem lengths were recorded
Main effect d.f. Probability*
in 8-day-old seedlings, five days after Trichoderma treatments were applied.
Values are means from three biological replicates ± standard deviations. Farm 1 0.06
Different letters indicate significant differences (Fisher test, P < 0.05). Treatment 2 0.00*
Year 1 0.84
Treatment Root length (cm) Stem length (cm)
Farm*Treatment 2 0.76
Farm*Year 1 0.48
Control 2.19 ± 0.64 a 2.78 ± 0.63 a
Treatment*Year 2 0.91
BCC1 1.56 ± 0.24b 2.66 ± 0.75 a
Farm*Treatment*Year 2 0.65
BCF2 1.54 ± 0.56b 2.82 ± 0.99 a
Error 108
BCF7 1.76 ± 0.50b 3.06 ± 0.94 a
Terms for Fisher comparisons Media (plants/m2)
Treatment T1 Control 4.59 ± 0.39 a
30.5 ton/ha, that is, an increase of 20.4% with respect to the control. T2 3.41 ± 0.34c
T3 3.61 ± 0.34b
The onion yield at the lowest dose of BCC1 (treatment T2) was also Model
significantly higher than the control, with an average of 29.02 ton/ha Mortality = 3.87 + 0.72 T1 Control − 0,46 T2 − 0,26 T3
and an increase of 18.4%.
Data represents the average of the replicates (n = 10) ± standard deviations.
Treatments tested were: T1 Control (untreated plants); T2 (plants treated with
3.5. Systemic defense triggered by T. asperellum BCC1 against S. cepivorum 1 × 106 conidia/mL of BCC1); and T3 (plants treated with 1 × 107 conidia/mL
SCM in onion plants of BCC1).
*For each effect, asterisks denote significant differences (P < 0.05). Values
The expression levels of defense marker genes in the central leaf of followed by different letters are significantly different according to the Fisher
onion plants were analyzed for the four treatments (T1, T2, T3 and T4) test (P < 0.05).
compared in the greenhouse assay at four time points (1, 2, 7, and 21
dpi with the pathogen). Table 5
The expression of AcPAL1 showed a different profile according to Generalized linear model for oniońs yield in field experiments.
whether the plant was challenged by T. asperellum BCC1 or S. cepivorum Main effect d.f. Probability*
(Fig. 1). This gene was strongly up-regulated by BCC1 at 1 and 7 dpi
comparing to the control but it was down-regulated at day 2 and 21 Farm 1 0.40
Treatment 2 0.00*
showing an undulating expression pattern. The expression level of
Year 1 0.03*
AcPAL1 in treatment T2 was 49.7- and 3.3-fold higher than in plants Farm*Treatment 2 1.00
receiving treatment T3 at days 1 and 7, respectively. When onion plants Farm*Year 1 0.57
were treated with the pathogen, the expression levels of AcPAL1 were Treatment*Year 2 0.71
significantly lower, compared to control treatment, at 1 (0.12-fold), 2 Farm*Treatment*Year 2 0.62
Error 108
(0.87-fold) and 21 dpi (0.30-fold). The combination of biocontrol agent
and pathogen caused a significant increase of expression of this gene Terms for Fisher comparisons Media (Kg/m2)
Year 2017 2.86 ± 0.36 a
when compared to plants treated solely with the pathogen at all times
2018 2.75 ± 0.37b
analyzed; however, the levels were significantly lower than those of Treatment T1 Control 2.45 ± 0.28c
plants treated with T. asperellum at day 1 (0.32-fold) but higher at 2 T2 2.90 ± 0.25b
(1.13-fold) and 21 (2.32-fold) dpi. T3 3.05 ± 0.27 a
Model
AcPR1 also showed a different expression pattern based on the in-
Yield = 2.80–0.35 T1 Control + 0.10 T2 + 0.25 T3 + 0.05 Year 2017–0.05 Year
oculated microbe. The highest levels of expression were observed at day 2018
7 for those plants treated with either T. asperellum or the combination of
biocontrol agent and pathogen (compared to control plants) (Fig. 1). Data represents the average of the replicates (n = 10) ± standard deviations.
The expression of AcPR1 in treatment T3 was 0.33-fold (at day 1), 0.87- Treatments tested were: T1 Control (untreated plants); T2 (plants treated with
fold (at day 2) and 0.30-fold (at day 21) lower, where only S. cepivorum 1 × 106 conidia/mL of BCC1); and T3 (plants treated with 1 × 106 conidia/mL
was applied to the plants, compared to untreated plants (T1), except at of BCC1).
day 7 where no significant differences were observed. Though later in *For each effect, asterisks denote significant differences (P < 0.05). Values
followed by different letters are significantly different according to the Fisher
time than the expression observed for AcPAL1, AcPR1 also showed an
test (P < 0.05).
undulating expression pattern starting from day 2 for those samples
treated only with BCC1.
Compared to control plants, T. asperellum caused a strong decrease
in the expression of AcLOX1 at day 1 (34.4-fold) (Fig. 1). Although this

Table 3
Effect of T. asperellum BCC1 treatment on the fitness of onion plants and biocontrol against S. cepivorum.
Treatment Root length (cm) Root dry weight (g) Stem length (cm) Canopy dry weight (g) Mortality percentage (%)

T1 Control 10.73 ± 1.61 a 0.10 ± 0.03 a 16.91 ± 1.78 a 0.17 ± 0.04 a 0

T2 9.18 ± 1.47b 0.12 ± 0.02 a 14.61 ± 1.85b 0.17 ± 0.02 a 0
T3 6.88 ± 0.99c 0.08 ± 0.01b 10.09 ± 2.34c 0.11 ± 0.03b 88
T4 9.32 ± 2.05 ab 0.11 ± 0.03 a 15.49 ± 1.73 ab 0.18 ± 0.03 a 14

Values are the mean of the replicates (n = 10, for growth parameters) and (n = 20, for mortality) with their corresponding standard deviation. Data were collected
15 dpi (for growth parameters) and 21 dpi (for mortality) with the pathogen. Treatments tested were: T1 control (untreated plants); T2 (plants treated with T.
asperellum BCC1); T3 (plants infested with S. cepivorum); and T4 (plants treated with BCC1 and S. cepivorum).
Values followed by different letters are significantly different according to the Fisher test (P < 0.05).

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W. Rivera-Méndez, et al. Biological Control 141 (2020) 104145

Fig. 1. Relative expression of AcPR1, AcPAL1, AcLOX1, and AcEIN3 in onion plants treated with T. asperellum, S. cepivorum or the combination of both fungal strains
(T. asperellum + S. cepivorum) in a 21-days time course study. Values correspond to relative measurements against the respective control condition (Untreated plants)
(2−ΔΔCT = 1) for each time point. Error bars represent standard deviations for five biological replicates at each time point. The actin gene (AcACT) was used as a
reference. Data was analyzed by one-way ANOVA and Tukey test. Different letters represent significant differences (P < 0.05).

down-regulation was moderately reduced from day 2, and the expres- U/g, respectively), while plants infected with S. cepivorum showed the
sion of this gene was never up-regulated at any of the other three time lowest activity (0.77 U/g and 6.40 U/g, respectively).
points analyzed. The expression of AcLOX1 seems to be more induced
by the pathogen, but only significantly up-regulation was detected at 7 4. Discussion
(3.6-fold) and 21 (6.3-fold) dpi.
The expression levels of AcEIN3 showed a continuing increment The potential of T. asperellum BCC1 as effective biocontrol agent of
from day 1 to 21 in those plants treated with T. asperellum though only S. cepivorum in onion plants was clearly proved in this study, as re-
was down-regulated at 1 dpi (0.25-fold) and up-regulated at 21 dpi (2- ductions in plant mortality due to white rot were observed in those
fold) when compared to control plants. No significant differences were plants treated with this strain in greenhouse and field experiments. We
observed among treatments at day 2 and 7 and only combination of should point out here the fact that the three T. asperellum strains as-
both fungal strains showed significant up-regulation of AcEIN3 at 21 sessed in this work were isolated from onion roots in production fields
dpi when compared to control plants (1.7-fold). from the district of Llano Grande in Cartago, Costa Rica (Alvarado-
The enzymatic PAL activity was calculated for all treatments Marchena and Rivera-Méndez, 2016), where field experiments de-
(Table 6). Only data collected at day 2 showed no significant differ- scribed in this study were taken place. This aspect should be considered
ences among treatments. The highest PAL activity was detected in a valuable asset for biocontrol applications as native isolates are better
plants from the T. asperellum treatment at 1 and 7 dpi (19.15 and 16.21 adapted to their local climate conditions and pathogenic targets than

Table 6
PAL activity (U/g of fresh tissue) measured in onion leaves in 4 treatments.
Treatment *dpi 1 dpi 2 dpi 7 dpi 21

Control 4.68 ± 0.18c 4.50 ± 0.15 a 4.58 ± 0.26c 4.52 ± 0.12 a

T. asperellum 19.15 ± 0.12 a 4.42 ± 0.34 a 16.21 ± 0.54 a 1.61 ± 0.08c
S. cepivorum 0.77 ± 0.00 d 4.25 ± 0.09 a 6.40 ± 0.33 d 2.70 ± 0.20b
T. asperellum + S. cepivorium 9.03 ± 0.20b 4.76 ± 0.12 a 12.30 ± 0.94b 1.87 ± 0.07c

Values are the mean of the replicas with their corresponding standard deviation.
Values followed by different letters are significantly different according to the Fisher test (P < 0.05).
*Days post-inoculation.
Regression equation of trans-cinnamic acid’ standard curve (y = 0.3836x−0.6501).

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W. Rivera-Méndez, et al. Biological Control 141 (2020) 104145

foreign isolates (Debbi et al., 2018). conditions were controlled, defense marker onion genes were analyzed
Based on the in vitro results, T. asperellum BCC1 was selected for in a 21-days time course study in order to know whether the reduction
biocontrol assays in onion plants in greenhouse and field trials. In fact, in onion plants mortality observed at 21 dpi with the pathogen in the
direct antagonism, to variable extent of performance among the three T. treatment T4, mirrored in changes of gene expression levels.
asperellum strains tested, was observed in both dual culture and cello- Plant defense responses to the T. asperullum root inoculation has
phane membrane assays. In any case, BCC1 showed the best perfor- been described as ISR type (Shoresh et al., 2005), but an increasingly
mance. The antagonism ability of Trichoderma species against S. cepi- number of studies are reporting SA-dependent defense responses trig-
vorum is well known (Hernández et al., 2011; Shalaby et al., 2013), but gered by Trichoderma spp. (Salas-Marina et al., 2011; Tucci et al.,
even though this pathogen is a main cause of important economic losses 2011). Our data show an undulating expression pattern for AcPAL1 and
in onion cultivars, yet white rot control has not been fully explored with AcPR1 genes, both SA-dependent defense markers, triggered by strain
Trichoderma strains. Some studies describe strategies to biocontrol S. BCC1 when applied alone to onion plants, as observed in Trichoderma
cepivorum in greenhouse or field trials in different climatic regions. parareesei-tomato interactions (Rubio et al., 2014). At this point, as
Torrés-Barragán et al. (1996) inoculated onion plants with the arbus- shown in Fig. 1, a down-regulation of JA/ET-dependent defense genes
cular mycorrhizal consortium Glomus sp. Zac-19 achieving significant (AcLOX1 and AcEIN3) was also observed at 1 day after application of
protection against S. cepivorum and 22% yield increment as compared strain BCC1 to onion plants. The lack of any relevant source of in-
with nonmycorrhizal controls in Mexico’s field assays. Similarly, onion formation regarding defenses regulation in onion plants against S. ce-
plants treated with Rhizophagus irregularis (formerly Glomus in- pivorum leads us to speculate that the response might be JA/ET-de-
traradices) showed less white rot incidence than those plants untreated pendent, similarly to that displayed against other necrotrophic
in onion organic fields in Ontario region (Canada) (Jaime et al., 2008). pathogens (Glazebrook, 2005). In this sense, the expression of AcLOX1
Antagonist bacterial isolates have been also described to reduce white was up-regulated at 7 and 21 dpi while there was not any significant up-
rot in onion plants compared with the untreated control in Egypt’s field regulation of SA-dependent genes compared to control plants. The use
trials (Elshahawy et al., 2018). The use of Trichoderma species to bio- of S. cepivorum sclerotia as inoculum could explain the delay in the JA-
control S. cepivorum in onion plants has been also described in field mediated systemic defense observed in plants at 1 dpi with the pa-
trials localized in Egypt, using T. koningii and T. harzianum (Shalaby thogen. As reported by Adams and Papavizas (1971), on autoclaved
et al., 2013), United Kingdom with T. viride (Clarkson et al., 2006; soil, hyphae emerge from the sclerotium within a period of about
Coventry et al., 2006), or Australia with T. koningii (Metcalf et al., 1–2 days. Considering that T. asperellum was applied to onion plants
2004). However, in countries like Costa Rica with soils rich in organic 7 days before the pathogen in the combined treatment (T4), it is quite
matter and where tropical climatic conditions easily promote the de- likely that there might be a SA-dependent priming based on the higher
velopment of white rot, there are not relevant studies that can con- AcPAL1 expression levels in this condition compared to what was ob-
tribute to implement the use of Trichoderma spp. as biocontrol agents served in plants treated with BCC1 alone. Thus, at day 21 when onion
themselves or as a tool within the integrated management control of plant mortality was recorded, the reduction observed in onion plants
this onion disease. from the treatment T4 could be explained by the synergic activity of the
Before field experiments were taken place, greenhouse assays were direct antagonism of T. asperellum against S. cepivorum observed in the
carried out with T. asperellum BCC1 to test its capabilities against S. in vitro studies and the plant systemic defenses induced by the BCC1
cepivorum in onion plants. Several studies had shown the potential of T. strain. An additional observation that corroborates the expression pat-
asperellum to biocontrol pathogens with diverse life styles in different tern of AcPAL1, was the PAL enzymatic activity data (Table 6) which
plants such as Rhizoctonia solani in cucumber (Trillas et al., 2006), showed similar undulating behavior to those detected in the expression
Pythium myriotylum in cocoya (Mbarga et al., 2012), Fusarium oxy- of this gene (Fig. 1). The transcription factor EIN3 activates ET-re-
sporum in tomato (Debbi et al., 2018), or Verticillium dahliae in olive sponsive genes (Contreras-Cornejo et al., 2015). ET is a key factor in the
cultivars (Carrero-Carrón et al., 2016). The amount of S. cepivorum regulation of root development processes, by inhibiting lateral root
sclerotia that was used as infective dose in the greenhouse assay, formation and elongation (Contreras-Cornejo et al., 2015; Hermosa
proved to be adequate for the outset of white rot development in onion et al., 2012). Then, the up-regulation of AcEIN3 observed in onion
seedlings because percentages of mortality of 88% were recorded in the plants for treatment T2 at 21 days, treated solely with T. asperellum,
treatment T3. As would be expected, onion plants infested with S. ce- might explain the absence of growth promotion. However, when in
pivorum (treatment T3) presented significantly less root and canopy dry combination with the pathogen, plant growth data were similar to those
biomass and smaller root and stem lengths. The mortality percentage of the control plants as well, being the AcEIN3 expression down-regu-
obtained for the treatment T4 was 14%, showing the efficiency of strain lated in comparison to treatment T2 but up-regulated when compared
BCC1 against S. cepivorum. Although lower root and stem length values to untreated plants.
were obtained when applying T. asperellum BCC1 to onion plants Results from controlled environment assays like greenhouses, rarely
(treatment T2), no significant differences were observed in dry weight translate to field evaluations due to fluctuating environmental condi-
values between these plants and those from the control condition. It is tions. However, assessment of T. asperellum BCC1 performance carried
important to point out the reduced root growth response of the onion out in two independent experiments in two consecutive season trials,
seedlings inoculated with any of the three T. asperellum strains tested. In provided conclusive results in terms of usefulness of this strain for fu-
this respect, it is worth mentioning that the ability to promote plant ture commercial purposes. Our data indicate no differences in the
growth is not a common characteristic extensible to all Trichoderma “Farm” factor what lead us to conclude that soil features (pH and soil
strains (Rubio et al., 2012). However, onion plants treated with the composition) are sufficiently similar to not be a key factor controlling S.
combination of strain BCC1 and S. cepivorum (treatment T4) did not cepivorum development or T. asperellum performance, and therefore
show worse phenotypic parameters (root and stem lengths) compared onion bulb production. However, year of planting revealed to be a
with control plants but decreased the onion plants mortality compared factor affecting yield, though not mortality, which may be related to
to that from treatment T3. The absence of significant differences in climatic conditions (temperature or humidity), despite no relevant
plant fitness between treatment T4 and treatments T1 and T2 may be variations were recorded in the closest weather station to the farms
indicative that activation of plant defenses is occurring in response to (data not shown). There was, however, a clear dose-dependent effect
the application of T. asperellum BCC1 alone. This outcome has been related to the application of T. asperellum BCC1 in the field trials. When
described earlier in Trichoderma (Alonso-Ramírez et al., 2014; Hermosa used to the highest concentration, white rot incidence and onion bulb
et al., 2013), and the activation of onion systemic defenses has been yield parameters were improved, regardless of farm localization and
investigated in this study. In the greenhouse assay where environmental harvesting seasonal variations. Likewise, higher concentrations of

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W. Rivera-Méndez, et al. Biological Control 141 (2020) 104145

conidia of T. asperellum seem to perform better against R. solani when Appendix A. Supplementary data
applied to cucumber seedlings (Trillas et al., 2006). And in rice plants
exposed to drought stress, increasing doses of T. harzianum applied to Supplementary data to this article can be found online at https://
the roots enhanced plant drought tolerance (Pandey et al., 2016). In doi.org/10.1016/j.biocontrol.2019.104145.
Costa Rica, the absence of tolerant or resistant onion cultivars to S.
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