agriculture

Article
Native Trichoderma Isolates from Soil and Rootstock to
Fusarium spp. Control and Growth Promotion of
Humulus lupulus L. Plantlets
Alejandra J. Porteous-Álvarez 1 , Alexia Fernández-Marcos 1 , Daniela Ramírez-Lozano 1 , Sara Mayo-Prieto 1 ,
Rosa E. Cardoza 2 , Santiago Gutiérrez 2 and Pedro A. Casquero 1, *

1 Grupo Universitario de Investigación en Ingeniería y Agricultura Sostenible (GUIIAS), Instituto de Medio

Ambiente, Recursos Naturales y Biodiversidad, Universidad de León, Avenida Portugal 41, 24071 León, Spain
2 Grupo Universitario de Investigación en Ingeniería y Agricultura Sostenible (GUIIAS), Área de Microbiología,
Escuela de Ingeniería Agraria y Forestal, Campus de Ponferrada, Universidad de León, Avenida Astorga s/n,
24400 Ponferrada, Spain
* Correspondence: [email protected]

Abstract: Fusarium genus is a wide host phytopathogen causing significant losses in multiple crops,
including hops. There is limited information on the sustainable management of Fusarium spp. in hop
fields. Trichoderma is an endophytic fungus used in agriculture as a biological control agent (BCA) and
as a plant growth promoter. It has been used to antagonize Fusarium spp. in other crops. The objective
of the current study was to identify indigenous hop field Trichoderma isolates with biocontrol and
hop growth promotion capabilities. Three isolates of Fusarium and eleven autochthonous Trichoderma
isolates collected from sustainable hop fields were evaluated in this work. Direct confrontation
tests (the physical interaction between the pathogen and BCA and their competition for space and
nutrient resources) and membrane tests (the capacity of the BCA to produce metabolites or enzymes
Citation: Porteous-Álvarez, A.J.; through a cellophane film and inhibit the development of the pathogen) assessed the antagonism of
Fernández-Marcos, A.; these Trichoderma isolates against Fusarium culmorum, F. sambucinum, and F. oxysporum. A bioassay
Ramírez-Lozano, D.; Mayo-Prieto, S.; with hop plantlets inoculated with a spore suspension of Trichoderma was performed to assess its
Cardoza, R.E.; Gutiérrez, S.; hop growth enhancement. T. hamatum (T311 and T324), T. virens T312, and T. gamsii T327 showed
Casquero, P.A. Native Trichoderma high growth inhibition of Fusarium spp. phytopathogens and high plant growth promotion. Native
Isolates from Soil and Rootstock to
Trichoderma isolates from sustainable hop-producing soils have great potential as BCAs and hop
Fusarium spp. Control and Growth
growth promoters.
Promotion of Humulus lupulus L.
Plantlets. Agriculture 2023, 13, 720.
Keywords: hops; biological control; antifungal activity; plant-growth promotion; soil microorganisms;
https://doi.org/10.3390/
sustainable agriculture; direct confrontation; dual culture; membrane assay
agriculture13030720

Academic Editors: Beata Janowska

and Maciej Bosiacki

Received: 14 February 2023

1. Introduction
Revised: 16 March 2023 Hops (Humulus lupulus L.) are long-lived, perennial, herbaceous, and dioecious climb-
Accepted: 16 March 2023 ing plants cultivated worldwide for the female flower cones used in the brewing and
Published: 21 March 2023 pharmaceutical industries. Hops are affected by several pests and diseases that can cause
significant economic losses [1]. The main pests and diseases affecting hops are mildew,
powdery mildew, Verticillium, Fusarium, aphids, and two-spotted spider mites. They all
cause significant reductions in the production and quality of cones, affecting farmers
Copyright: © 2023 by the authors.
economically [2].
Licensee MDPI, Basel, Switzerland.
This article is an open access article
There is a concern to control the main pests and diseases affecting hop fields and help
distributed under the terms and
reduce economic losses. Detailed research has been done on the management of aphids on
conditions of the Creative Commons
hops [1–5]. Several research groups are currently studying the biology and management of
Attribution (CC BY) license (https:// powdery mildew [6–10]. There is a rising concern among hop farmers about the presence
creativecommons.org/licenses/by/ of Fusarium spp. in hop fields and the ways to control it. Fusarium spp. in hops has been
4.0/). described as a problem in Europe [11,12], the USA [13], and Brazil [14].

Agriculture 2023, 13, 720. https://doi.org/10.3390/agriculture13030720 https://www.mdpi.com/journal/agriculture

Agriculture 2023, 13, 720 2 of 15

Fusarium spp. is a wide host phytopathogen causing significant losses in multiple

crops, including hops. It survives in the soil or on living plant material and accesses the
plant through wounds or natural openings in the plant tissue at ground level. Fusarium
sambucinum Brondeau can cause Fusarium canker or wilt and Fusarium cone tip blight [15].
F. oxysporum Schltdl. and F. culmorum (Wm. G. Sm.) Sacc. can cause hop wilting [16]. In
Fusarium cone tip blight, a necrosis at the tip of the cone is observed. In Fusarium canker
and wilt, the affected area of the stem swells while, near the crown, the stem narrows
again. Infected branches can be covered with white-pink or red-brown mycelium during
the growth or sporulation of the fungus on the stem surface [12,17,18].
There is limited information about the management of these infections with sustainable
solutions in hop fields. The need to find new sustainable and environment-friendly solu-
tions against phytopathogens in different crops has led to the search for biocontrol agents
(BCA) belonging to genera such as Bacillus, Beauveria, Fusarium, Pseudomonas, Streptomyces,
or Trichoderma [19–22]. Many research reports show possible sustainable solutions to
control Fusarium spp. using Trichoderma spp. in vitro or in crops including melon or wild
apple [23–28], but no similar studies have been conducted to control Fusarium spp. on hops.
Trichoderma spp. is an important endophytic fungal genus, a fast-growing secondary
opportunistic invader, avirulent [29,30], and beneficial to agriculture because it pro-
tects crops against phytopathogens like Rhizoctonia solani, Fusarium spp., Sclerotinia, and
Alternaria [26,31–33] and promotes plant growth and development, as reported by many
authors over time [24,34–36]. The benefits to agriculture are associated with the production
of antibiotics and plant growth regulators (PGRs) [37] and the induction of systemic resis-
tance in the plants [38]. Trichoderma can promote plant growth due to different mechanisms
like mobilizing nutrients in the soil, producing siderophores, solubilizing phosphorus or
synthesizing PGRs [24,34,35,39]. The increment of the plant biomass has been associated to
with synthesis of auxin-derivative compounds like indole-3-acetic acid (IAA) or other PGRs
like gibberellic acid (GA3 ) [34,40–43]. Moreover, indigenous microorganisms co-evolve
with a particular crop over time and adapt to environmental changes. Isolates collected
from a given crop may provide better biocontrol capabilities than isolates collected from a
different plant species [44]. It is essential to develop rational management against pests and
diseases in our crops. To do so, select autochthonous Trichoderma isolates with beneficial
characteristics for the plant, such as plant growth promotion and biocontrol of pathogens.
The aim of this study was to identify autochthonous isolates of Trichoderma adapted
to hop-producing fields that show biocontrol and plant growth promotion abilities and
to evaluate their antagonism against native Fusarium isolates and their hop plant growth
promotion activity.

2. Materials and Methods

2.1. Trichoderma and Fusarium Isolates and Culture Collection
The present assay was performed with three Fusarium isolates collected in the hop-
producing area of San Román de la Vega, León (Spain), from fields presenting Fusarium-like
symptoms. The Trichoderma isolates used in the study were collected from four sustainable
hop-producing fields (Table 1) [45]. All isolates were stored in the collection of pathogens
and antagonists at the ‘Laboratorio de Diagnóstico de Plagas y Enfermedades Vegetales’
(LDPEV), Universidad de León, Spain.
The Nucleospin Plant II kit (Macherey-Nagel, Düren, Germany) was used to extract the
genomic DNA from 100 mg of each fungal isolate, following the manufacturer’s protocol
for fungi. The resulting extracts were eluted in 50 µL of sterile water, and a NanoDrop
ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used to estimate
the DNA concentration. Solutions of 50 µL containing 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl2, 1.5 U of DreamTaq DNA polymerase (Thermo Scientific), 200 nM
for each dNTP, 400 nM for each primer, and 50 ng of DNA were used to amplify the
sequences. For the identification of the Trichoderma isolates analyzed in the present work, a
fragment corresponding to the ITS region was amplified using the oligonucleotides ITS5 (50 -
Agriculture 2023, 13, 720 3 of 15

GGAAGTAAAAGTCGTAACAAGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ). For

the identification of Fusarium spp. in the present work, an internal fragment of the gene tef1
was amplified using the oligonucleotides EF1-728F (50 -CATCGAGAAGTTCGAGAAGG-30 )
and EF1-986R (50 -TACTTGAAGGAACCCTTACC-30 ). The PCR products were purified
using the NucleoSpinExtract II kit (Machery-Nagel, Düren, Germany), and sequenced
afterwards using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,
Foster City, CA, USA) with an automatic capilar sequencer ABI 3130xl (Applied Biosystems).
All the steps were performed in accordance with the manufacturer’s instructions. In order
to identify the fungal isolates, the sequences obtained were analyzed and compared with
those in the NCBI Genbank database (National Center for Biotechnology Information,
http://www.ncbi.nlm.nih.gov, accessed on 17 March 2023) using the BLAST tool (http://
www.ncbi.nlm.nih.gov/BLAST, accessed on 17 March 2023). The generated sequences were
deposited at DDBJ/ENA/Genebank. Accession numbers are specified in Table 1.

Table 1. Trichoderma and Fusarium isolates collected from hop-producing plots in León (Spain).

ID Treatment Identified as % Identity Origin Location Accession Number

T311 T. hamatum 1 >99% Soil Gavilanes de Órbigo OQ590011
T324 T. hamatum 1 >99% Rootstock San Román de la Vega OQ589841
T312 T. virens 1 >99% Soil Gavilanes de Órbigo OQ590010
T317 T. virens 1 >99% Rootstock Nistal OQ589506
T327 T. gamsii 1 >99% Soil Seisón de la Vega OQ589844
T316 T. rossicum 1 >99% Rootstock Gavilanes de Órbigo OQ589503
T328 T. rossicum 1 >99% Soil Gavilanes de Órbigo OQ589861
T329 T. harzianum 1 >99% Soil San Román de la Vega OQ589868
T314 T. spirale 1 >99% Soil Nistal OQ589492
T319 T. spirale 1 >99% Rootstock Nistal OQ589706
T323 T. brevicompactum 1 >99% Soil San Román de la Vega OQ589712
F076 F. culmorum 2 >99% Soil San Román de la Vega OQ625436
F103 F. oxysporum 2 >99% Soil San Román de la Vega OQ632904
F079 F. sambucinum 2 >99% Rootstock San Román de la Vega OQ632903
1 The isolates were identified by ITS regions. 2 The isolates were identified by EF regions.

2.2. In Vitro Antifungal Assays-Direct Confrontation

The direct confrontation assay evaluated the in vitro physical interaction between
the pathogen and BCA and their competition for space and nutrient resources [32]. The
inoculum of pathogen and BCA was obtained from the growing edge of 7 day cultures
growing on potato-dextrose-agar medium (PDA, Sigma-Aldrich, St. Louis, MO, USA) at
25 ◦ C and collected using a 6 mm diameter cork borer. The plugs were sown on the same
Petri plate with PDA medium, with a 55 mm distance between plugs. Each treatment was
represented by 4 replicates. Growth diameters of the pathogen were measured after five
days, taking two measures of the colony: r1, the radium of the colony growing towards
the BCA, and r2, the radium growing away from the BCA; the latter served as a control
for the growth of the pathogen against the growth of the colony closer to Trichoderma. The
percentage of growth inhibition caused by Trichoderma was calculated using Equation (1):

Growth inhibition (%) = (r2 − r1)/r2·100. (1)

2.3. In Vitro Antifungal Assays-Membrane Assay

The membrane assay evaluated the capacity of the BCA to produce metabolites or
enzymes through a cellophane film and inhibit the development of the pathogen without
direct contact [32]. Inoculum plugs were collected as described above. On a Petri plate
with PDA medium, a cellophane film was placed in contact with the medium, and a 6 mm
Trichoderma plug was placed on the center of the membrane and left to grow for 48 h. After
that time, the film was removed, and a 6 mm plug of the Fusarium isolate was placed on the
 2.3. In Vitro Antifungal Assays-Membrane Assay
The membrane assay evaluated the capacity of the BCA to produce metabolites or
enzymes through a cellophane film and inhibit the development of the pathogen without
Agriculture 2023, 13, 720 direct contact [32]. Inoculum plugs were collected as described above. On a Petri plate4 of 15
with PDA medium, a cellophane film was placed in contact with the medium, and a 6 mm
Trichoderma plug was placed on the center of the membrane and left to grow for 48 h. After
that time, the film was removed, and a 6 mm plug of the Fusarium isolate was placed on
center of the plate. The growth of the fungi was evaluated five and eight days later, taking
the center of the plate. The growth of the fungi was evaluated five and eight days later,
two orthogonal measurements
taking two orthogonal of theofgrowth
measurements diameters.
the growth Four
diameters. replicates
Four were
replicates wereevaluated
eval-
per treatment. Plates with no Trichoderma
uated per treatment. Plates with no Trichoderma served as a control. The data are presented as a
served as a control. The data are presented
percentage of pathogen
as a percentage growth
of pathogen inhibition
growth compared
inhibition comparedto to
thethe
growth
growthononcontrol
controlplates.
plates.

2.4.
2.4.In
In Vivo
Vivo Assay-Trichoderma GrowthPromotion
Assay-Trichoderma Growth PromotionActivity
Activity
The
Theinin vivo
vivo assay
assay evaluated
evaluated thethegrowth
growthpromotion
promotionofofH. H.lupulus
lupulusL.L.‘Columbus’
‘Columbus’plant-plantlets
by Trichoderma isolates under controlled climatic conditions. The bioassay
lets by Trichoderma isolates under controlled climatic conditions. The bioassay was per- was performed
with
formed one-month-old
with one-month-oldplantlets obtained
plantlets from from
obtained aerialaerial
partsparts
of ‘Columbus’
of ‘Columbus’ cultivar.
cultivar.After
one
After one month, the plantlets formed their own root systems. Plantlets were grown in
month, the plantlets formed their own root systems. Plantlets were grown 100 mL
in 100
volume pots with non-autoclaved peat substrate (Exclusive Substrate,
mL volume pots with non-autoclaved peat substrate (Exclusive Substrate, Gebr. Brill Sub- Gebr. Brill Substrate
GmbH & Co. &
strate GmbH KG,
Co.Georgsdorf,
KG, Georgsdorf, Germany)
Germany)and and
werewere
watered
wateredweekly.
weekly.
The bioassay was performed in a climate chamber at 25 ◦ C and 70% relative humidity
The bioassay was performed in a climate chamber at 25 °C and 70% relative humidity
(RH)
(RH)during
duringthethe day
day and
and 15 °C◦ C and
and 75%
75%RH RHduring
duringthethenight,
night,with
witha photoperiod
a photoperiod of of 16:8 h
16:8
day:night.
h day:night.
Ten plants
Ten plants per treatment
treatmentwere wereselected,
selected, and their
and initial
their development
initial development waswas measured.
measured.
Allplants
All plantspresented
presented oneone string at thethe beginning
beginningof ofthe
theassay,
assay,with
withaasimilar
similaraverage
average num-
number
ofber of nodes
nodes (5.4 +(5.4
1.9)+in
1.9)
allin all treatments
treatments at theatbeginning
the beginning
of theoftrial.
the trial.
Figure Figure 1 shows
1 shows an
an example
ofexample of two plantlets
two plantlets with thewith thedevelopment
initial initial development
of oneofstring
one string and to
and five fivesix six pairs
to pairs of
of leaves,
leaves,
or nodes.or nodes.

Figure 1. Hop plantlets with one string and five–six nodes per plant.
Figure 1. Hop plantlets with one string and five–six nodes per plant.

Plantlets were inoculated with 5 mL of each Trichoderma spore solution (1 × 107 sp/mL).
Plants inoculated with 5 mL of water were used as control plants. They were kept in the
growing chamber under the climate conditions described above for two months. Plant
growth was evaluated one and two months after inoculation by tallying the number of
strings and the number of nodes per string, counting from the base of the plant to the last
pair of leaves distinct from the tip bud.
Two months after the inoculation, soil samples were collected from the surface and
the soil surrounding the rootstock of the plants. Soil samples were spread on Rose Bengal
chloramphenicol agar medium (Conda Laboratory, Torrejón de Ardoz, Madrid, Spain) and
incubated at 25 ◦ C in the dark to confirm Koch´s postulates, as Trichoderma growth was
observed in the culture media with soils inoculated with Trichoderma and there was no
growth of Trichoderma on control pots.
 last pair of leaves distinct from the tip bud.
Two months after the inoculation, soil samples were collected from the surface and
the soil surrounding the rootstock of the plants. Soil samples were spread on Rose Bengal
chloramphenicol agar medium (Conda Laboratory, Torrejón de Ardoz, Madrid, Spain)
and incubated at 25 °C in the dark to confirm Koch´s postulates, as Trichoderma growth
Agriculture 2023, 13, 720 5 of 15
was observed in the culture media with soils inoculated with Trichoderma and there was
no growth of Trichoderma on control pots.

2.5.
2.5.Statistical
StatisticalAnalysis
Analysis
Alldata
All datawere
werecompared
compared by analysis
analysisofofvariance
variance(ANOVA)
(ANOVA) and compared
and by by
compared Fisher’s
Fisher’s
least significant difference (LSD) (p < 0.05) post hoc tests using SPSS (IBM SPSS Statistics
least significant difference (LSD) (p < 0.05) post hoc tests using SPSS (IBM SPSS Statistics
forWindows,
for Windows,Version
Version26.0,
26.0, Armonk,
Armonk, NY,NY, USA).
USA).

3.3.Results
Results
3.1.
3.1.InInVitro
VitroAntifungal
Antifungal Assay-Direct Confrontation
Assay-Direct Confrontation
Direct
Directconfrontation
confrontation assays evaluatedthe
assays evaluated thecompetition
competition forfor
thethe media
media resources
resources andand
spaceofofthe
space theTrichoderma
Trichoderma isolates
isolates against
againstFusarium
Fusariumspp.
spp.InIn
thethe
current study,
current thethe
study, isolates
isolates
performeddifferently
performed differentlywith
with each pathogen
pathogenisolate
isolate(Figure
(Figure2).2).

(a) (b)
Figure 2. Direct confrontation of F. culmorum with (a) T. hamatum T311 (65.8% of growth inhibition)
Figure 2. Direct confrontation of F. culmorum with (a) T. hamatum T311 (65.8% of growth inhibition)
and (b) T. rossicum T328 (49.7% of growth inhibition). In the image, Fusarium sp. is on the left, and
and (b) T. rossicum T328 (49.7% of growth inhibition). In the image, Fusarium sp. is on the left, and
Trichoderma spp. is on the right.
Trichoderma spp. is on the right.
Figure 3 represents the mean percentages of growth inhibition of the Fusarium spp.
Figure
assayed. 3 represents
Regarding the mean
the direct percentages
confrontation of growth
results, inhibition
the isolates of the Fusarium
of T. hamatum (T311 andspp.
assayed. Regarding the direct confrontation results, the isolates of
T324) showed the highest values of growth inhibition against F. culmorum (65.8% T. hamatum (T311
andand
T324)
66.3%)showed
and F.the highest (47.5%
oxysporum values and
of growth
50.6%),inhibition
respectively.against
AgainstF. culmorum (65.8%T.and
F. sambucinum,
66.3%)
hamatum F. oxysporum
andT311 exhibited(47.5% and 50.6%),
the highest respectively.
inhibition percentage,Against F. sambucinum,
46.3%, without T. hamatum
significant dif-
T311 exhibited
ferences with T.the highest
hamatum inhibition
T324, percentage,
36.5%. The isolates of46.3%, without
the species significant
T. virens differences
(T312 and T317)
with T. hamatum
showed T324, 36.5%.
high inhibition The isolates
rates against the Fusarium isolates,T.but
of the species virens
these(T312
valuesand T317)
were showed
lower.
high inhibition rates against the Fusarium isolates, but these values were lower than the
performance of the T. hamatum isolates. T. virens isolates performed similarly against all
Fusarium isolates, having no significant differences among each other. T. virens T317 had no
significant differences with T. hamatum in the growth inhibition of F. culmorum (61.0%) and
F. sambucinum (35.8%). T. gamsii T327 showed 43.2% and 42.8% growth inhibition against
F. sambucinum and F. oxysporum, respectively, with no significant differences to T. hamatum
(T311 and T324). The inhibition percentage shown by T. gamsii T327 against F. culmorum
(54.0%), although high, was significantly lower compared to the performance of T. hamatum
(T311 and T324). T. rossicum (T316 and T328) showed the lowest values on growth inhibition
of Fusarium spp. compared to the rest of the Trichoderma isolates. T. rossicum T328 presented
the lowest inhibition percentages, 13.4% and 11.2% against F. sambucinum and F. oxysporum,
respectively. T. rossicum T328 showed 49.7% growth inhibition in F. culmorum. T. harzianum
T329 caused a 52.0% growth inhibition of F. culmorum without significant differences with
T. virens T312 and T. gamsii T327. Against F. oxysporum, T. harzianum T329 had a 30.0%
growth inhibition, without significant differences with T. virens (T312 and T317). Against
Agriculture 2023, 13, 720 6 of 15

F. sambucinum, T. harzianum T329 showed a high inhibition percentage (36.6%) without

Agriculture 2023, 13, x FOR PEER REVIEW 6 of
significant differences to T. hamatum (T311 and T324).

(a)

(b)

(c)

Figure 3. Growth inhibition Figurepercentages

3. Growth inhibition
of thepercentages
Trichoderma of the Trichoderma
isolates isolates
against (a)against (a) F. culmorum, (b)
F. culmorum,
sambucinum, and (c) F. oxysporum in vitro by direct confrontation. Mean values ± SE. Columns with
(b) F. sambucinum, and (c) F.the
oxysporum in vitro by direct confrontation. Mean values ± SE. Columns
same chart with different letters indicate significant differences (Fisher’s LSD p < 0.05).
within the same chart with different letters indicate significant differences (Fisher’s LSD p < 0.05).

The isolates of T. spirale (T314 and T319) showed significantly similar inhibition per-
centages for F. culmorum (51.7% and 54.3%) but interacted differently against the other
Fusarium species. T. spirale T314 had high inhibition percentages against F. sambucinum
(40.7%) without significant differences with T. hamatum (T311 and T324), and T. spirale T319
showed a higher inhibition percentage against F. oxysporum (34.6%) without significant
Agriculture 2023, 13, 720 7 of 15

differences with T. virens (T312 and T317) and T. gamsii T327. T. brevicompactum T323 pre-
sented a 53.1% growth inhibition of F. culmorum and 30.8% and 34.0% growth inhibition
against F. sambucinum and F. oxysporum, respectively, without significant differences to
T. rossicum T316.
Overall, the isolates of T. hamatum, (T311 and T324) showed a high inhibition percent-
age of the three Fusarium isolates. T. virens (T312 and T317) and T. gamsii T327 presented
high inhibition values but lower than the T. hamatum isolates, with significant differences
against F. oxysporum in the case of T. virens and F. culmorum in T. gamsii T327. T. rossicum
(T316 and T328) showed significant differences with the top isolates but still presented high
inhibition percentages for F. culmorum. T. harzianum T329 had adequate growth inhibition
rates against F. culmorum, F. sambucinum, and F. oxysporum.

3.2. In Vitro Antifungal Assay-Membrane Assay

The membrane assays evaluated the capacity of each Trichoderma isolate to produce
metabolites and enzymes with antibiosis activity under laboratory conditions. Figure 4a
shows how Trichoderma spp. grew on the cellophane membrane without physical con-
tact with the PDA medium. Figure 4b shows how F. sambucinum grew naturally on
Agriculture 2023, 13, x FOR PEER REVIEW 8 of 17 of
PDA medium in a negative control plate, and Figure 4c shows the inhibited growth
F. sambucinum on PDA after removing the cellophane with the T. gamsii T327 colony.

(a) (b) (c)

Figure 4. (a) Trichoderma spp. growing on the membrane without physical contact to the medium,
Figure 4. (a) Trichoderma spp. growing on the membrane without physical contact to the medium,
(b) F. sambucinum growing on a negative control on potato-dextrose-agar medium (PDA, Sigma-
F. sambucinum
(b)Aldrich, growing on a negative control on potato-dextrose-agar medium (PDA, Sigma-
St. Louis, MO, USA), and (c) F. sambucinum growing on PDA after removing the membrane
Aldrich, St. Louis,
with T. gamsii T327 MO, USA),
(74.9% F. sambucinum growing on PDA after removing the membrane
and (c)inhibition).
of growth
with T. gamsii T327 (74.9% of growth inhibition).
Figure 5 represents the growth inhibition in the membrane assays of all the Tricho-
Figure
derma 5 represents
isolates the growth
against Fusarium spp.inhibition in theshowed
T. gamsii T327 membrane goodassays of all the
performance Trichoderma
in produc-
isolates Fusarium spp. T. gamsii
tion of metabolites with antifungal activity against all the pathogens assayed and an out- of
against T327 showed good performance in production
metabolites with antifungal
standing growth inhibition activity
percentage against
for F.all the pathogens
sambucinum assayed
(74.9%). and an(T311
T. hamatum outstanding
and
growth inhibitiongreater
T324) presented percentage
antibiosis F. sambucinum
for against F. culmorum (53.3% T.
(74.9%). andhamatum
41.8%) and (T311 and T324)
F. sambuci-
presented
num (63.9% greater antibiosis
and 64.1%) but low against F. culmorum
antibiosis (53.3% and
against F. oxysporum 41.8%)
(21.8% andand F. sambucinum
17.2%). T. virens
(63.9%
(T312 and T317)
64.1%) but low
showed highantibiosis against F.values
growth inhibition oxysporum
against(21.8% and 17.2%).
F. sambucinum (64.4%T.andvirens
67.5%)
(T312 andand F. oxysporum
T317) showed (44.9% and 42.8%)
high growth but lower
inhibition inhibition
values againstrates against F. culmorum
F. sambucinum (64.4% and
(28.7%and
67.5%) andF.23.0%). T. spirale
oxysporum (44.9%(T314andand T319)but
42.8%) hadlower
their inhibition
highest inhibition rates against
rates against F.
F. culmorum
sambucinum
(28.7% (38.1% and
and 23.0%). 35.0%)(T314
T. spirale without and significant
T319) had differences between
their highest T. spiralerates
inhibition T319 against
and
F. T. harzianum T329.
sambucinum (38.1%Against F. oxysporum,
and 35.0%) without T. significant
spirale (T314differences
and T319) presented
between T. a low inhi-
spirale T319
bition percentage (19.0% and 18.0%), with no significant differences
and T. harzianum T329. Against F. oxysporum, T. spirale (T314 and T319) presented a low to T. hamatum (T311
and T324).
inhibition T. harzianum
percentage T329and
(19.0% showed
18.0%),lowwith
inhibition percentages
no significant againstto
differences allT.the Fusarium
hamatum (T311
isolates assayed, with the highest value being 29.2% in F. sambucinum.
and T324). T. harzianum T329 showed low inhibition percentages against all the Fusarium T. brevicompactum
T323 had
isolates a high inhibition
assayed, rate in F.value
with the highest sambucinum (58.5%)inwithout
being 29.2% significantT.differences
F. sambucinum. to
brevicompactum
T. hamatum and T. spirale and a 45.0% inhibition rate against F. culmorum
T323 had a high inhibition rate in F. sambucinum (58.5%) without significant differences to and F. oxysporum.
The isolates of T. rossicum (T316 and T328) had the lowest values against all the Fusarium
isolates, all under 20.0% growth inhibition, with significant differences with the rest of the
Trichoderma isolates.
Altogether, F. culmorum and F. oxysporum showed a lower influence by Trichoderma
metabolites, whereas F. sambucinum was more susceptible, as the percentages of inhibition
Agriculture 2023, 13, 720 8 of 15

T. hamatum and T. spirale and a 45.0% inhibition rate against F. culmorum and F. oxysporum.
The isolates of T. rossicum (T316 and T328) had the lowest values against all the Fusarium
Agriculture 2023, 13, x FOR PEER REVIEW 9 of
isolates, all under 20.0% growth inhibition, with significant differences with the rest of the
Trichoderma isolates.

(a)

(b)

(c)

Figure 5. Growth inhibition percentages of the Trichoderma isolates against (a) F. culmorum, (b) F.
Figure 5. Growth inhibition percentages of the Trichoderma isolates against (a) F. culmorum,
sambucinum, and (c) F. oxysporum in antifungal membrane assays. Mean values ± SE. Columns
(b) F. sambucinum, and (c) F.within
oxysporum in chart
the same antifungal membrane
with different letters assays.
indicate Mean values
significant ± SE. (Fisher’s
differences Columns LSD, p < 0.05
within the same chart with different letters indicate significant differences (Fisher’s LSD, p < 0.05).

Altogether, F. culmorum and F. oxysporum showed a lower influence by Trichoderma

metabolites, whereas F. sambucinum was more susceptible, as the percentages of inhibition
had higher values. In general, the metabolite secretion of T. hamatum (T311 and T324) and
Agriculture 2023, 13, 720 9 of 15

T. gamsii (T327) showed greater antibiosis against F. culmorum and F. sambucinum. The
metabolites secreted by T. virens (T312 and T317) presented high growth inhibition values
against F. sambucinum and F. oxysporum. T. harzianum T329 had low antagonism against the
Fusarium isolates assayed, and the isolates of T. rossicum (T316 and T328) showed the lowest
Agriculture 2023, 13, x FOR PEER REVIEW
antibiosis against all the Fusarium isolates. T. rossicum (T316 and T328) and T.10harzianum
of 17

T329 showed the lowest antibiosis against all the Fusarium isolates.

3.3.
3.3.In
In Vivo
Vivo Assays-Trichoderma GrowthPromotion
Assays-Trichoderma Growth Promotion
The
The bioassays
bioassays inin the
theclimate
climatechamber
chamber evaluated
evaluated thethe growth
growth promotion
promotion features
features of the
of the
Trichoderma
Trichoderma isolates
isolates on ‘Columbus’
‘Columbus’cultivar
cultivarplantlets
plantlets under
under controlled
controlled conditions.
conditions. Plants
Plants
used
usedin inthe
thetrial
trialpresented
presented aa significantly similar development
significantly similar developmenton onthe
theday
dayofofinoculation;
inoculation; all
plants had had
all plants the same number
the same numberof strings andand
of strings a similar number
a similar of nodes
number distributed
of nodes evenly
distributed
evenly throughout
throughout the treatments.
the treatments. Therefore,
Therefore, treatments
treatments were were compared
compared amongamong
themthem
without
withoutconsideration
further further consideration in the following
in the following measurements.
measurements. FigureFigure
6 shows6 shows hop plant-
hop plantlets in the
lets in the climate chamber
climate chamber during the assay. during the assay.

Figure 6. Hop plantlets in the climate chamber during the growth promotion assay.
Figure 6. Hop plantlets in the climate chamber during the growth promotion assay.

Figure 77 represents
Figure represents thethemean
meanvalues
valuesofof
the development
the development measures taken
measures one one
taken and and
two two
months after the treatments. After one month, plants in the Trichoderma treatments not
months after the treatments. After one month, plants in the Trichoderma treatments did did not
significantly increase
significantly increase their
theirdevelopment
developmentover overthe controls
the controls(CC), except
(CC), for T.
except forrossicum T328T328
T. rossicum
thatsignificantly
that significantly increased
increasedthe thenumber
numberofof nodes
nodes plant
perper (Figure
plant 7b) 7b)
(Figure andandT. virens T312T312
T. virens
that significantly increased the number of nodes per string (Figure 7c). After
that significantly increased the number of nodes per string (Figure 7c). After one month, one month,
T.T.gamsii
gamsii T327-treated plants, presented the highest number of strings, showing four times
T327-treated plants, presented the highest number of strings, showing four times
the initial number of strings. Treatments with T. rossicum T328, T. gamsii T327, and T. spi-
the initial number of strings. Treatments with T. rossicum T328, T. gamsii T327, and T. spirale
rale T319 showed the biggest enhancement in the number of nodes on the hop plantlets
T319 showed the biggest enhancement in the number of nodes on the hop plantlets after
after one month. T. virens T312 caused a significant enhancement in the number of nodes
one month. T. virens T312 caused a significant enhancement in the number of nodes per
per string compared to CC.
string compared to CC.
After two months from the inoculation, the CC plants showed lower development
After two
compared months
to most from the
Trichoderma inoculation,
treatments. the CC(T311
T. hamatum plants showed
and lower
T324), T. virensdevelopment
T312, T.
compared to most Trichoderma treatments. T. hamatum (T311 and
gamsii T327, T. rossicum T328, and T. harzianum T329—treated plantlets presented T324), T. avirens
signif-T312,
T.icant
gamsii T327, T. rossicum T328, and T. harzianum T329—treated
developmental enhancement, as shown by higher numbers of strings (over three plantlets presented a
significant developmental enhancement, as shown by higher numbers
strings per plant) and the number of nodes (more than ten nodes per plant) compared to of strings (over three
strings
CC (1.2per plant)
strings andand3.8 the number
nodes). of nodes
Treatments (more
with than ten
T. hamatum nodes
T324, per plant)
T. gamsii T327, andcompared
T.
to CC (1.2T328
rossicum strings and more
showed 3.8 nodes).
than fourTreatments
nodes perwith T. significantly
string, hamatum T324,higherT. gamsii
than CC T327,
(2.3 and
nodes per string).
T. brevicompactum T323 and T. spirale T319 showed low growth promotion abilities in
hop plantlets without significant differences from CC plants. The isolates of T. rossicum
Agriculture 2023, 13, 720 10 of 15
Agriculture 2023, 13, x FOR PEER REVIEW 11 of 17

T. rossicum T328 showed more than four nodes per string, significantly higher than CC
T316 and T. virens T317 presented the lowest values of plant growth development, with
(2.3 nodes per string). no significant differences to CC plants regarding strings and nodes per plant.

(a)

(b)

(c)

Figure 7. Growth development measures: (a) strings per plant, (b) nodes per plant, and (c) nodes
Figure 7. Growth development
per stringmeasures:
(down). Mean(a)values
strings
(n = per
10) ±plant,
SE. The(b) nodes
mean valuesper
afterplant, and are
one month (c)represented
nodes in
per string (down). Meanstripes,
values (nafter
and = 10)two±months
SE. The mean
in solid values
colors. afterwith
Columns onedifferent
month lower-case
are represented
letters indicate
in stripes, and after two months in solid colors. Columns with different lower-case letters indicate
significant differences after one month (Fisher’s LSD p < 0.05). Columns with different capital letters
indicate significant differences after two months (Fisher’s LSD, p < 0.05).

T. brevicompactum T323 and T. spirale T319 showed low growth promotion abilities in
hop plantlets without significant differences from CC plants. The isolates of T. rossicum
T316 and T. virens T317 presented the lowest values of plant growth development, with no
significant differences to CC plants regarding strings and nodes per plant.
Agriculture 2023, 13, 720 11 of 15

4. Discussion
Trichoderma is widely used as a BCA and is reported to show antagonistic effects
against phytopathogenic microorganisms but also beneficial effects on the host plants, such
as the production of plant growth metabolites, nutrient uptake, or induction of defense
mechanisms on the host plant [30,37]. The selection of a Trichoderma isolate has been
commonly based on their in vitro antagonistic activity as a purpose for its application as
a BCA, but it is interesting to select them also by their ability to interact and promote
plant growth. In the current study, different autochthonous Trichoderma isolates collected
from soil samples of sustainable hop fields interacted in vitro with the phytopathogens
F. sambucinum, F. culmorum, and F. oxysporum and in vivo with ‘Columbus’ cultivar plants.
Two methods were explored to evaluate the antagonistic potential of the Trichoderma
isolates. Firstly, through direct confrontation, Trichoderma spp. competed for space and
nutrients, inhibiting the development of the pathogen [25,32,46]. Secondly, membrane
assays, metabolites, and enzymes produced by the Trichoderma isolates are released through
a cellophane membrane into the growing medium with no physical contact with the fungi,
and their activity against the pathogens can be evaluated [32,47,48].
Growth of all Fusarium spp. analyzed was inhibited in direct confrontation assays
with Trichoderma spp. The isolates of T. hamatum (T311 and T324) showed the best response
against all Fusarium isolates. T. virens (T312 and T317) and T. gamsii (T327) presented high
inhibition values against all Fusarium isolates but significantly lower than the T. hamatum
isolates. T. harzianum T329 showed good direct confrontation skills against F. sambucinum.
These data agree with those of other authors showing the ability of T. harzianum and
T. hamatum to inhibit F. oxysporum growth in vitro [24,25]. T. rossicum T328 showed the
lowest values, however, and caused nearly 50% growth inhibition in F. culmorum. These
results agreed with those of Ji et al. [27], where T. rossicum inhibited Fusarium sp. growth
in vitro by 41% in wild apples.
In the membrane assays, T. hamatum T311 and T. gamsii T327 were the isolates with
higher antagonism by metabolite production against F. culmorum and F. sambucinum. These
results agreed with previous findings describing the in vitro inhibition of R. solani growth
by T. gamsii metabolites [48,49] and F. oxysporum [26]. T. virens (T312 and T317) showed
antifungal activity against F. sambucinum and F. oxysporum. This species has proven its
inhibitory capacity of Fusarium spp. and other pathogens such as R. solani, Botrytis cinerea,
S. sclerotiorum, etc. [50]. T. harzianum T329 showed low inhibition against the isolates of
Fusarium assayed, which contrasts with previous works with other isolates of the same
species [26,51–53]. Vinale et al. [54] observed that the production of secondary metabolites
by T. harzianum could be affected by the interaction with the phytopathogen. The low
interaction of this isolate of T. harzianum against Fusarium spp. could be related to the
fact that the metabolites produced in interaction with these pathogens have no antibiosis
effect against them, as the antagonism is not species-dependent [49], or to the inability of
producing secondary metabolites under laboratory conditions [55,56]. T. rossicum T316 and
T328 showed very low inhibition percentages against all Fusarium spp. analyzed, indicating
that they may not produce antibiotic compounds under laboratory conditions.
Regarding plant-growth promotion activity, the isolates T. hamatum (T311 and T324),
T. virens T312, T. gamsii T327, T. rossicum T328, and T. harzianum T329 had a good overall
performance inducing hop growth in the early stages of development. In agreement with
our findings, T. hamatum, T. harzianum, T. virens, and T. gamsii showed good plant promotion
performances reported by different authors in muskmelon, tomato, and Arabidopsis, among
others [23,24,27,34,35,57]. T. harzianum induced a significant increase in root density, plant
height, and number of buds when applied to hemp plants [36]. T. rossicum was used together
with T. harzianum as a biofertilizer to promote plant growth in wild apple plants [27].
T. hamatum isolates showed favorable results in growth promotion and antagonism assays,
in accordance with Martínez-Medina et al. [24] in melons.
The isolates T. rossicum (T316 and T328) presented similar performances in the in vitro
assays but had very different outcomes in plant growth, similarly to the isolates T. virens
Agriculture 2023, 13, 720 12 of 15

(T312 and T317). This could be explained by the origin of the isolates. T. virens T312 and
T. rossicum T328 were collected from soil samples, whereas T. virens T317 and T. rossicum
T316 were collected from rootstock samples. Mayo-Prieto et al. [48] claimed that Trichoderma
isolates from soil samples may have better biocontrol abilities than isolates from other
sources. Calvet et al. [58] observed that the inoculation of T. aureoviride alone had no effect
on plant growth, contrary to the inoculation of Trichoderma with the arbuscular mycorrhizal
fungus Glomus mosseae. Similar observations were made by Siddiqui and Mahmood [59]
with T. harzianum. This symbiotic effect on Trichoderma-plant interaction may be due
to the alteration of the rhizosphere by the arbuscular mycorrhizal fungi, making their
presence essential to obtain agronomic benefits from the plant growth promoter fungi [60].
Regarding T. rossicum T328 and its results in the antagonism assays and plant promotion,
we may consider its lack of abilities to produce antibiosis under laboratory conditions [55],
and the Trichoderma-plant-pathogen interaction may be considered for further assays to
determine its biocontrol capacities.
The results obtained in this work identify the ability of Trichoderma spp. to con-
trol Fusarium spp. and promote plant growth. Hoyos-Carbajal et al. [39] claimed that
Trichoderma spp. was able to produce auxin-type PGRs like IAA. Contreras-Cornejo et al. [34]
observed that plant biomass enhancement was due to the auxin-dependent mechanisms
triggered by T. viride in Arabidopsis. Nieto-Jacobo et al. [43] observed similar results in
Arabidopsis with different Trichoderma species. Bader et al. [42] observed that the Trichoderma
isolates that presented a higher production of IAA also showed high antagonism against
F. oxysporum in tomatoes. The present work evaluated the ability of the BCA to control and
promote plant growth. There are good perspectives on some of the isolates assayed, al-
though we consider that further studies must be conducted to characterize the mechanisms
of the selected isolates and to understand the effects of the BCA not just in plantlets but
also in adult plants and cone yield.
The present work evaluates the interaction between a single pathogen and a single
BCA; however, every isolate exhibited different modes of action that may have comple-
mentary effects if used in a mixture or combination. The work of Duffy et al. [61] demon-
strated that the combination of T. koningii and P. fluorescens showed enhanced biocontrol
in wheat than T. koningii alone. Ji et al. [33] observed that the combination of T. rossicum
and T. harzianum promoted wild apple growth. However, before any further consideration
on Trichoderma combinations, it is important to know how they interact among themselves
and with the existing non-pathogenic microbiome, due to the complexity of rhizosphere
interactions [62]. Further research should be conducted to better understand the implica-
tions of Trichoderma against hop pathogens.
Thus, the results of these experiments showed the possible use of Trichoderma spp.
as a BCA in hop plants. It opens a new line of research to evaluate different species of
Trichoderma in hops, identify volatile compounds and PGRs produced that benefit the hop
plant cultivation in plantlets and adult plants, and observe the impact this BCA may have
on the hop cone yield.

5. Conclusions
To summarize, T. hamatum (T311 and T324) and T. gamsii T327 showed control re-
sponses against Fusarium spp. and growth promotion of hop plantlets. T. hamatum T311
exhibited the highest control of F. culmorum through metabolite secretion, T. gamsii T327
against F. sambucinum, and T. virens T312 against F. oxysporum. T. rossicum T328 and
T. harzianum T329 showed little antagonism in vitro but exhibited plant growth promotion
features. Some Trichoderma isolates collected from sustainable hop-producing soils can act as
sustainable solutions to evaluate new disease control strategies and induce hop promotion.
Trichoderma native isolates collected from sustainable hop-producing soils compete for
space and nutrients and mycoparasitize Fusarium isolates, showing an inhibited develop-
ment in the pathogen. Trichoderma native isolates from sustainable hop-producing soils have
great potential as BCAs and hop growth promoters. The native isolates of T. hamatum (T311
Agriculture 2023, 13, 720 13 of 15

and T324), T. virens T312, and T. gamsii T327, all from sustainable hop-producing soils, can
serve to develop a sustainable solution to control diseases and enhance hop development.

Author Contributions: Conceptualization, P.A.C. and S.M.-P.; methodology, A.F.-M. and A.J.P.-Á.;
software, A.J.P.-Á.; validation, A.F.-M. and S.M.-P.; formal analysis, A.J.P.-Á. and A.F.-M.; investi-
gation, A.F.-M., D.R.-L. and A.J.P.-Á.; resources, S.G. and P.A.C.; data curation, A.J.P.-Á.; writing—
original draft preparation, A.J.P.-Á.; writing—review and editing, A.J.P.-Á., S.M.-P., R.E.C. and P.A.C.;
visualization, A.J.P.-Á., D.R.-L. and P.A.C.; supervision, S.M.-P., S.G. and P.A.C.; project administra-
tion, S.G. and P.A.C.; funding acquisition, P.A.C. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the Ministerio de Universidades (Spain), grant number
FPU19/03650 to A.J.P.-Á., and the Ministerio de Agricultura, Pesca y Alimentación (Spain), Quality
Hops Operational Group, Innovations in the cultivation of hops in Spain to improve the sustainability
of farms (2019/00179/001).
Institutional Review Board Statement: Not applicable.
Acknowledgments: The authors thank the field workers in charge of the hop farms and laboratory
technicians for their help and collaboration in the necessary tasks.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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