Journal of

Fungi
Review
Microbial Biological Control of Fungi Associated with
Grapevine Trunk Diseases: A Review of Strain Diversity,
Modes of Action, and Advantages and Limits of
Current Strategies
Ouiza Mesguida 1,2, *, Rana Haidar 1 , Amira Yacoub 1 , Assia Dreux-Zigha 2 , Jean-Yves Berthon 2 ,
Rémy Guyoneaud 1 , Eléonore Attard 1, * and Patrice Rey 1, *

1 E2S UPPA, CNRS, IPREM, Universite de Pau et des Pays de l’Adour, 64000 Pau, France
2 GreenCell: Biopôle Clermont-Limagne, 63360 Saint Beauzire, France
* Correspondence: [email protected] (O.M.); [email protected] (E.A.);
[email protected] (P.R.)

Abstract: Grapevine trunk diseases (GTDs) are currently among the most important health challenges
for viticulture in the world. Esca, Botryosphaeria dieback, and Eutypa dieback are the most current
GTDs caused by fungi in mature vineyards. Their incidence has increased over the last two decades,
mainly after the ban of sodium arsenate, carbendazim, and benomyl in the early 2000s. Since then,
considerable efforts have been made to find alternative approaches to manage these diseases and
limit their propagation. Biocontrol is a sustainable approach to fight against GTD-associated fungi
and several microbiological control agents have been tested against at least one of the pathogens
involved in these diseases. In this review, we provide an overview of the pathogens responsible, the
various potential biocontrol microorganisms selected and used, and their origins, mechanisms of
action, and efficiency in various experiments carried out in vitro, in greenhouses, and/or in vineyards.
Lastly, we discuss the advantages and limitations of these approaches to protect grapevines against
Citation: Mesguida, O.; Haidar, R.;
Yacoub, A.; Dreux-Zigha, A.; Berthon,
GTDs, as well as the future perspectives for their improvement.
J.-Y.; Guyoneaud, R.; Attard, E.; Rey,
P. Microbial Biological Control of Keywords: Botryosphaeria dieback; Esca; Eutypa dieback; biological interactions; plant microbiome;
Fungi Associated with Grapevine microbial interactions
Trunk Diseases: A Review of Strain
Diversity, Modes of Action, and
Advantages and Limits of Current
Strategies. J. Fungi 2023, 9, 638. 1. Introduction
https://doi.org/10.3390/jof9060638 As was the case for earlier grapevine health crises at the end of the 19th century with
Academic Editor: Katrina phylloxera, powdery, and downy mildews, the viticulture sector is now confronted with
Maria Ramonell vast upheavals, such as climate change, associated with high societal expectations for an
environmentally friendly viticulture, as well as the major crisis of grapevine trunk disease
Received: 2 May 2023 (GTD) epidemics. With regard to GTDs, which re-emerged in the late 1990s, it took a mere
Revised: 23 May 2023
two decades for Esca, the most frequent one, to become a subject of major concern for many
Accepted: 25 May 2023
viticulture regions in Europe and worldwide. GTDs represent a group of vascular diseases
Published: 31 May 2023
caused by fungi affecting grapevine wood, mainly through pruning wounds, and inhabiting
the xylem cells in the woody tissue [1,2]. The colonization of this tissue leads to a decline in
the plant host because of a loss of the xylem function and subsequent decrease in hydraulic
Copyright: © 2023 by the authors.
conductivity, causing significant necrosis and decay with time, which ultimately lead to
Licensee MDPI, Basel, Switzerland. foliar symptoms and grapevine death [3–5]. Esca, Botryosphaeria dieback, and Eutypa
This article is an open access article dieback are the most frequent on mature grapevines; they decrease vineyard longevity,
distributed under the terms and thereby affecting wine quality and causing huge economic losses throughout the viticulture
conditions of the Creative Commons sector [6,7].
Attribution (CC BY) license (https:// Until now, up to 133 fungal species belonging to 34 genera have been associated with
creativecommons.org/licenses/by/ GTDs in the literature [8], most of them growing slowly, found alone or together in the
4.0/). same plant, for several years [2,9]. After the infection onset of pathogenic fungi, it takes a

J. Fungi 2023, 9, 638. https://doi.org/10.3390/jof9060638 https://www.mdpi.com/journal/jof

J. Fungi 2023, 9, 638 2 of 37

long time, usually years, before the appearance of the first foliar symptoms [10]. When the
first foliar symptoms occur, they are often linked to the development of rot necrosis in the
grapevine trunk or cordons [4]. However, GTDs leave symptoms expressed inconsistently
from year to year on individual grapevines [1,2]. The main pathogenic fungi involved
in these diseases are Neofusicoccum parvum, Diplodia seriata, and Lasiodiplodia theobromae
for Botryosphaeria dieback, Phaeomoniella chlamydospore, Phaeoacremonium minimum, and
Fomitiporia mediterranea for Esca, and Eutypa lata for Eutypa dieback [1,2].
Vitis vinifera cultivars display different levels of tolerance and react with defense
mechanisms to cope with the vascular pathogens involved in GTDs [5,10]. The tolerance or
susceptibility of grapevine cultivars to vascular fungal pathogens has not yet been fully
explained but (i) the small xylem vessel diameter [5,11], (ii) the high levels of phenolic
compounds and lignin in the wood, and (iii) the early and rapid induction of defense-
related genes with greater accumulation of stilbene compounds and pathogenesis-related
proteins [12] have been reported to explain the differences in susceptibility across cultivars.
Previously, sodium arsenate, carbendazim, and benomyl were used to control GTDs.
However, the use of these products was banned in early 2000s because of their toxicity
toward humans and the environment. To mitigate the economic losses due to GTDs,
as no effective control treatments currently exist since, several strategies based on the
employment of biological agents, chemical compounds, and cultural practices are used
alone or in combination to limit GTD incidence [1,2].
Several methods of control, including cultural practices, chemicals, and biological
control products have been tested against grapevine trunk diseases [1]. They can be
grouped into preventive and curative methods [6,13]. As for the preventive methods,
measures are recommended before, during, and after planting [6,14]. Before planting, it is
recommended to use controlled mother vineyards of good quality with limited age, and
to avoid the most GTD-susceptible cultivars in the most fertile soils [14]. At planting, it
is important to avoid the long immersions of roots in water [14]. After planting and in
the vineyards, according to whether the target vines are already affected or not, several
prophylactic methods are applied to control GTDs [13,14]. Among them, it is important to
take care of the correct training of the trunks by avoiding the short-pruned wounds that
can cause drying zones inside the trunk [14]. In addition, the method and time of pruning
can affect the susceptibility of wounds to pathogenic fungi [13,14]. Guyot-Poussard is
the most used pruning system as it ensures an optimal flow of sap [13]. However, the
use of such a system is still not fully understood or justified, due to a lack of evidence of
efficiency in relevant experimental trials [13]. Lecomte et al. (2012) recommended a pruning
period in late winter, particularly to prevent Eutypa [15]. The protection of pruning wounds
using natural or chemical products is another method used to limit wound infection by
pathogens [8,16].
As for the curative methods in GTD-affected vineyards, “remedial surgery” is applied
to eliminate by pruning the symptomatic woody parts from affected vines (cordons and/or
trunks), until healthy wood is left [8,13]. If the majority of the vine trunk exhibits internal
symptoms of GTD, the technique used is the trunk renewal [8]. In some countries, trunk
surgery or “curettage” is another practice used to remove the rotten tissues in the trunk of
GTD-affected vines using electric handsaws [13].
All these management strategies may help to prevent GTDs. However, GTD control is
still challenging and problematic because of the rarity of efficient strategies, as well as the
complexity of the diseases with a high diversity of biotic and abiotic factors involved in the
different disease stages. Wound protection remains the most effective technique for limiting
the dissipation of pathogens. Thus, the search for effective strategies for the protection of
wounds, notably via biological control, is essential for the management of GTDs.
Biological control is a promising sustainable alternative approach to fight GTD-causing
fungal pathogens; during the last decade, about 1600 microorganisms with potential bio-
control activities (MBCAs) were investigated. Most of the studies were carried out with
bacterial and fungal strains, but a few oomycetes and actinobacteria were also used to bio-
 limiting the dissipation of pathogens. Thus, the search for effective strategies for the pro-
tection of wounds, notably via biological control, is essential for the management of GTDs.
Biological control is a promising sustainable alternative approach to fight GTD-caus-
ing fungal pathogens; during the last decade, about 1600 microorganisms with potential
J. Fungi 2023, 9, 638 biocontrol activities (MBCAs) were investigated. Most of the studies were carried out3with of 37
bacterial and fungal strains, but a few oomycetes and actinobacteria were also used to
biocontrol GTD pathogens [1]. These MBCAs stopped and/or destroyed the pathogenic
fungi through
control a number[1].
GTD pathogens of direct or indirect
These MBCAs mechanisms
stopped and/or of action [17,18].
destroyed Direct interac-
the pathogenic fungi
tions occur when there is competition for spaces and nutrients, the production
through a number of direct or indirect mechanisms of action [17,18]. Direct interactions of sidero-
phoreswhen
occur and hydrolytic enzymes, parasitism,
there is competition or antibiosis
for spaces and nutrients,[18]. On the otherofhand,
the production indirect
siderophores
and hydrolytic enzymes, parasitism, or antibiosis [18]. On the other hand, indirectfurther
mechanisms mainly consist of the induction of defense mechanisms in the plant mech-
to its colonization
anisms by a of
mainly consist biocontrol agent of
the induction (Figure 1). mechanisms in the plant further to its
defense
colonization by a biocontrol agent (Figure 1).

Figure 1. The key mechanisms of action of MBCAs assessed toward GTD-associated fungi. (1) Com-
Figure 1. The key mechanisms of action of MBCAs assessed toward GTD-associated fungi.
petition for space and nutrients between the MBCAs and the pathogen(s). In terms of nutrients, they
(1) Competition for space and nutrients between the MBCAs and the pathogen(s). In terms of
compete for micronutrients such as manganese, specific growth substances (i.e., amino acids), or
nutrients,
stimulantsthey compete for micronutrients
for germination (i.e., fatty acids).such
(2)asProduction
manganese, ofspecific growththat
siderophores substances
mediate(i.e.,
ironamino
com-
acids), or stimulants for germination (i.e., fatty acids). (2) Production of
petition and lead to reduced pathogen populations. (3) Production of hydrolytic enzymes that siderophores that mediate
per-
iron competition
meabilize and lead
and degrade theto reducedcell
pathogen pathogen populations.
wall (e.g., chitinase, (3) Production
glucanase, of hydrolytic
protease, enzymes
and cellulase…),
that permeabilize
causing cell death.and degrade theconsisting
(4) Parasitism, pathogen of cella wall
direct(e.g., chitinase,
attack glucanase,
of the pathogen byprotease,
the MBCA,andwhich
cellu-
lase. . . ), causing cell death. (4) Parasitism, consisting of a direct attack of the pathogen by the MBCA,
leads to the invasion and destruction of the pathogen. (5) Antibiosis, whereby the MBCAs produce
inhibitory
which leads metabolites or antibiotics
to the invasion that affectofthe
and destruction growth
the or the(5)
pathogen. metabolic activity
Antibiosis, of thethe
whereby plant path-
MBCAs
ogen. (6) Induced systematic resistance (ISR), whereby the MBCAs induce a plant
produce inhibitory metabolites or antibiotics that affect the growth or the metabolic activity of the defense response
similar to that induced after pathogen infection. Created with BioRender.com. Accessed on 20 Feb-
plant pathogen. (6) Induced systematic resistance (ISR), whereby the MBCAs induce a plant defense
ruary 2023.
response similar to that induced after pathogen infection. Created with BioRender.com. Accessed on
20 February 2023.
In this review, we provide an overview of the current knowledge about the microbi-
ological control
In this agents
review, used to an
we provide manage the main
overview of thepathogens involved about
current knowledge in GTDs.
the We high-
microbio-
light the empirical evidence on their potential efficiency and mechanism of action,
logical control agents used to manage the main pathogens involved in GTDs. We highlight and we
the empirical
outline evidence
the current on their
practices usedpotential
to manage efficiency
GTDs. and mechanism of action, and we
outline the current practices used to manage GTDs.

2. Biocontrol of Botryosphaeria Dieback

Botryosphaeria dieback is associated with several Botryosphaeriaceae species [2].
Around 26 different Botryosphaeriaceae taxa have been found in vineyards of several coun-
tries, but Neofusicoccum parvum, Diplodia seriata, Phaeoacremonium minimum, Lasiodiplodia theo-
bromae, Neofusicoccum australe, Neofusicoccum luteum and Botryosphaeria dothidea are the most
widespread and aggressive species associated with Botryosphaeria dieback [8,19–21]. They
cause shoot dieback, cankers, central necroses in wood, and/or grapevine dieback [22]. The
species within the genera Lasiodiplodia and Neofusicoccum are the fastest wood-colonizing
fungi [8,23].
J. Fungi 2023, 9, 638 4 of 37

Most of this section is dedicated to biocontrol of the three most studied Botryosphaeri-
aceae species, N. parvum, D. seratia, and L. theobromae, before ending with three papers
aimed at controlling N. australe or other Botryosphaeria dieback-associated fungi.

2.1. Biological Control of Neofusicoccum parvum

Regarding the pathogenicity of N. parvum, Pitt et al. (2013) reported that N. parvum is
one of the most virulent species associated with Botryosphaeria dieback, according to the
lesion length they produce on mature wood tissue [24]. During grapevine colonization, N.
parvum produces phytotoxic metabolites with low molecular weight, including (3R,4R)-( )-
4-hydroxy-mellein and its stereoisomer (3R,4S)-( )-4-hydroxy-mellein, ( )-(R)-mellein,
( )-terremutin, isosclerone, and tyrosol [25–28]. N. parvum also produces hydrophilic high-
molecular-weight exopolysaccharides with phytotoxic activities [25,26]. The phytotoxic
activities of these secondary metabolites have been elucidated, but their contribution to the
development of Botryosphaeria dieback symptoms is still unknown [27,28]. In addition,
it was reported that N. parvum produces extracellular proteins with enzymatic activities
involved in wood degradation such as hydrolases and oxidoreductases, which are likely
involved in cell-wall and lignin degradation [19]; they are considered virulent factors
responsible for pathogenicity [29].
Therefore, N. parvum is a key target to develop biocontrol products against Botryosphaeria
dieback. From this perspective, multiple fungus isolates have been evaluated for their
potential antagonistic activity against N. parvum such as Chaetomium sp., Cladosporium sp.,
Clonostachys rosea, Epicoccum spp., Epicoccum nigrum, Fusarium proliferatum, Purpureocillium
lilacinum, and Trichoderma spp. [1,30–32].

2.1.1. Biocontrol Using Trichoderma

Trichoderma spp.—N. parvum Interactions In Vitro
Trichoderma spp. showed high efficiency in wound protection against all GTD pathogens [28].
In 2020, Úrbez-Torres et al. tested in dual culture the antagonistic capabilities of 16 Tri-
choderma strains isolated from southern Italy against N. parvum. The highest percentage
inhibition of radial mycelial growth of N. parvum (74.3%) was obtained with the strain T.
koningiopsis PARC1024 that was isolated from Prunus persica [33]. Ten Trichoderma strains
were assessed for their antagonistic activity against N. parvum in vitro by Kotze et al. (2011);
two T. atroviride strains, coded USPP-T1 and USPP-T2, were isolated from V. vinifera in South
Africa, and there were eight commercial strains, i.e., three T. atroviride, coded AG3, AG5,
and AG8, and five T. harzianum, coded AG2, AG11, Agss28, Biotricho, and Eco77. All these
strains were able to overgrow the pathogens in vitro, and microscopic observations revealed
coiling or hyphal adhesion between the pathogen’s hyphae and the Trichoderma’s hyphae
(for strains AG3, AG5, AG11, USPP-T1, and USPP-T2) [34]. Kotze et al. (2011), suggested
mycoparasitism and competition for nutrients as mechanisms of action of these strains.
As for Trichoderma endophytes, the growth of N. parvum was significantly reduced
by about 80% using the endophytic strains T. atroviride (ATCC 74058) and T. harzianum
(ATCC 26799) on PDA. These two strains did not show any ability to outcompete this
pathogen on carbon or nitrogen sources, although T. harzianum had some niche overlap
with it [35]. Blundell et al. (2021) reported for the first time the efficiency of a grapevine sap
T. hamatum strain against N. parvum in vitro.
Recently, Kovács et al. (2021) examined the potential ability of two Trichoderma strains
isolated from cordon wood of the grapevine cultivar Furmint in Hungary to inhibit the
growth of N. parvum in vitro. The two isolates, identified as T. afroharzianum (TR04) and
T. simmonsii (TR05), showed high potential against this pathogen with biocontrol indices
of 95.19% and 90%, respectively. In dual culture, T. afroharzianum (TR04) and T. simmonsii
(TR05) overgrew the N. parvum colony, and their hyphae coiled and penetrated the ones of
the pathogens [31], which is a sign of mycoparasitism [36]. According to Pollard-Flamand
et al. (2022), 26 Trichoderma strains obtained from grapevine roots and the basal end of either
rootstock or self-rooted vines in British Columbia significantly inhibited the growth of N.
J. Fungi 2023, 9, 638 5 of 37

parvum in vitro. They belonged to seven species: T. asperelloides, T. atroviride, T. harzianum, T.

koningii, T. tomentosum, T. canadense, and T. viticola.

Trichoderma spp.—N. parvum Interactions In Planta

Under controlled conditions, on detached grapevine cane, one strain of T. atroviride,
one of T. paratroviride, and one of T. guizhouense effectively protected pruning wounds
against N. parvum for at least 21 days after treatment. T. paratroviride PARC1012 gave
a mean percentage of disease control greater than 90% only 1 day after treatment [33].
These authors hypothesized that the mode of action of Trichoderma spp. to protect pruning
wounds against N. parvum was competition for nutrients and space [33]. T. canadense, T.
viticola, T. harzianum, T. atroviride, T. asperelloides, and T. koningii were tested for their ability
to protect pruning wounds against N. parvum on detached cane assays under controlled
greenhouse conditions; all these strains were effective in protecting from the pathogenic
infection. Higher protection was obtained with the strains of T. asperelloides, T. atroviride,
and T. canadense, which provided 96–100% pruning wound protection for up to 21 days
after treatment [37].
In the vineyard, Kotze et al. (2011) evaluated the ability of 10 Trichoderma strains
sprayed on fresh pruning wounds in a South African vineyard. The results showed that the
strain T. atroviride USPP-T1 was the most efficient and reduced the incidence of N. parvum
by 80% when challenged 7 days after treatment by the pathogen [34].

2.1.2. Biocontrol Using Other Fungal Genera

In vitro, it was reported that Chaetomium sp. showed a significant reduction in N. parvum
growth in vitro [1,30] with a reduction by 86.75% after 21 days of dual culture on an agar
medium based on 200 g/L grapevine dormant cutting. These authors hypothesized that
the antagonistic activity of Chaetomium sp. against N. parvum was due to mycoparasitism
because it grew slowly and inhibited the pathogen until colony contact [30].
Cladosporium sp. obtained from sprouts of asymptomatic grapevine showed interesting
antagonist activity against N. parvum in the agar medium based on 200 g/L grapevine
dormant cutting. The growth of this pathogenic fungi was reduced by 34.26% in the
presence of Cladosporium sp. Its antagonistic activity was presumably due to two modes of
action: (i) antibiosis, since, in the coculture test, N. parvum growth terminated before direct
contact of the two colonies; (ii) the highest sporulation rate of Cladosporium sp., because,
in the dual-culture assay, the growth inhibition of N. parvum was related to the strong
sporulation of Cladosporium sp. and not to the rapid growth of its mycelium [30].
Epicoccum is a genus of Ascomycetes associated with the wood mycobiome of grapevines
with known biocontrol potential [38,39]. The strain Epicoccum nigrum R29.1, isolated from
the root of asymptomatic grapevine, was tested in vitro for its potential to inhibit the growth
of N. parvum, but no significant results were obtained [30].
The antagonistic activity of C. rosea against GTD-associated fungi has been investigated
recently [30,40]. Silva-Valderrama et al. (2021) studied three C. rosea strains in vitro and
in planta and suggested that C. rosea strains were promising biocontrol agents of GTD
pathogens. These C. rosea strains were isolated from asymptomatic Cabernet Sauvignon
and Chardonnay commercial vineyards in Chile. Two strains (C. rosea CoR2.15 and C. rosea
R36.1) were root endophytes, and one (C. rosea CoS3/4.24) was from the rhizosphere. When
these three strains were tested to control N. parvum in vitro, all of them inhibited over 98% of
pathogen growth on day 21 [30]. For two strains, there was direct contact between colonies,
and light microscope observation revealed hyphal coiling in the confronting zone of the
two mycelia, suggesting mycoparasitism as the mechanism of action. As for the third strain,
the N. parvum growth inhibition was without physical contact between the colonies, and its
growth was terminated in correspondence with the halo surrounding it [30]. There were
changes in the colony morphology of N. parvum, which turned into several flat independent
colonies with undulate margins in contact with secondary metabolites secreted by C. rosea
J. Fungi 2023, 9, 638 6 of 37

CoS3/4.24. In this case, it was suggested that the antagonistic activity of C. rosea was due
to the secreted antibiotic compound [30].
Purpureocillium lilacinum has antibacterial, antimalarial, antifungal, antiviral, and anti-
tumor activities, and it is also known for its toxic activities against phytopathogens, notably
Phytophthora capsica [41]. Recently, it was shown that P. lilacinum inhibited N. parvum in vitro
without evident physical contact between colonies, suggesting the secretion of secondary
metabolites [30].
Aureobasidium spp. isolated from grapevine canes and sap, as well as one strain
identified as A. pullulans, were inefficient against N. parvum when tested in vitro [42]. It
was shown that Lecanicillium lecanii (ATCC 46578) caused a reduction by 15% in N. parvum
growth in vitro, acting via direct antagonism. According to Wallis et al. (2021), it also
effectively some niche overlap with this pathogen.
In planta, Silva-Valderrama et al. (2021) performed an assay on annual detached shoots
to study the antagonistic activity of the three C. rosea strains mentioned above against
N. parvum. These strains had a good efficiency to inhibit N. parvum, with the endophytic
isolates (C. rosea CoR2.15 and C. rosea R36.1) showing a better inhibition of N. parvum in
grapevine woody shoots compared with the rhizospheric strain, C. rosea CoS3/4.24 [30].
Mondello et al. (2019) reported that, under greenhouse-controlled conditions, and in a
vineyard planted in 1997 in France with the Mourvèdre/3309 cultivar, Fusarium prolifera-
tum limited the development of N. parvum by priming plant defense response when this
pathogen was inoculated 7 days after treatment with the Fusarium [32].

2.1.3. Biocontrol Using Oomycetes

Pythium oligandrum is a rhizospheric, nonpathogenic oomycete that colonizes the root
system of many cultivated plant species, including grapevine [43,44]. The biological control
exerted by P. oligandrum is due to direct effects on the pathogens (i.e., via mycoparasitism
or antibiosis) and/or indirect effects by resistance induction and growth promotion of the
plant [45]. Daraignes et al. (2018) carried out a 2 year study demonstrating that P. oligan-
drum root colonization reduced the wood necrosis length caused by N. parvum in young,
grafted cuttings of Cabernet Sauvignon grapevine. Because the pathogen and P. oligandrum
colonized different plant organs and never came into contact, these authors assumed that
induction of the grapevine defense system was the mode of action of P. oligandrum. Under
similar greenhouse conditions, Yacoub et al. (2020) showed that root system colonization
by P. oligandrum was associated with the reduction in wood necrosis caused by N. parvum
in rooted cuttings of Cabernet Sauvignon grapevine [43]. These authors also studied the
expression of 62 genes involved in grapevine defense pathways and observed that the prim-
ing of certain genes occurred at early stages, 14 days after the pathogen inoculation. They
highlighted the upregulation of PR protein genes, e.g., PR1, a marker of the salicylic acid
pathway and antifungal activity, GLU and PR2 encoding 6-1,3-glucanase, PR4bis encoding
chitinase, and PR14 involved in the defense signaling pathway, as well as those involved in
cell-wall reinforcement (e.g., CAD and CAGT), the indole signaling pathway (e.g., HSR203J,
CHORM, and CHORS2), and hormone signaling pathways (e.g., EDS1, ACO1, SAMT1,
and WRKY2), and genes affecting the salicylic acid pathway (e.g., SAMT1 and EDS1). No
synergetic effect between P. oligandrum and a bacterial strain with potential biocontrol
activity, Pantoea agglomerans or Bacillus pumilus, was observed by Daraignes et al. (2018) in
their greenhouse experiment.

2.1.4. Biocontrol Using Bacteria

The bacterial biocontrol of Botryosphaeria dieback pathogens has been explored,
mainly targeting the pathogen N. parvum [1]. Strains belonging to Bacillus spp. isolated from
healthy vineyards were highly efficient in protecting pruning wounds against various GTD
pathogens in vitro, in the nursery/greenhouse, and even in the field [28,42]. Bacillus spp.
antagonized GTD pathogens via various modes of actions such as antibiosis, competition for
nutrients, activation of plant defense system, and detoxification of pathogen toxins [28,42].
J. Fungi 2023, 9, 638 7 of 37

Strains of Bacillus subtilis were described as promising plant protectors against many fungal
pathogens, including in grapevine against pathogens causing wood staining [46–48].
In an in vitro study with B. subtilis, Kotze et al. (2011) used a strain isolated from
the woody tissue of grapevine wood arms (Chenin Blanc cultivar) that expressed Eutypa
dieback symptoms in South Africa. In the inhibition zone between N. parvum colonies and
the B. subtilis strain, light swelling and malformation of the pathogen hyphae was observed,
likely due to antibiotic molecule production by the bacteria [34].
In greenhouse studies with B. subtilis on young grapevine plants, a B. subtilis strain
(coded PTA-271) isolated from the rhizosphere of healthy Chardonnay grapevines in Cham-
pagne (France) was able to use both indirect and direct mechanisms to protect grapevine
cuttings against N. parvum. Furthermore, the inoculation of grapevine cuttings with this
PTA-271 bacterial strain in the soil for 1 month, then with the pathogen, significantly en-
hanced systemic grapevine immunity by priming the expression of PR2, encoding enzymes
involved in abscisic acid biosynthesis [28]. This B. subtilis strain also triggered the expres-
sion of salicylic acid- and jasmonic acid-responsive genes involved in the detoxification
process of key aggressive phytotoxins produced by N. parvum, i.e., ( )-terremutin and
(R)-mellein. Because this detoxifying process is more active in a nutrient-rich medium for
( )-terremutin, but not for (R)-mellein, Trotel-Aziz et al. (2019) suggested that (R)-mellein
was probably metabolized directly, while ( )-terremutin required a co-substrate to be co-
metabolized. This bacterium also acted directly on the pathogen, as shown by its fungistatic
effect on N. parvum hyphae [28].
With respect to assays in the field, in South African vineyards, after 8 months of
treatment with B. subtilis, a reduction by 16.5% in the incidence of this pathogen was
observed by Kotze et al. (2011); however, this decrease was not significantly different from
observations on unprotected wounds.
Regarding other in vitro assays with Bacillus and other bacteria species, Blundell et al. (2021)
investigated the in vitro ability of Bacillus velezensis to inhibit N. parvum. Given that they
observed a small zone of inhibition in the dual-culture assay and in the volatile assay
corresponding to 10% growth inhibition, they concluded that a volatile antibiotic was
produced [42]. The endophytic Bacillus sp. 3R1, Brevibacillus sp. 3Y41, and Bacillus sp.
3R4 strains, isolated from a 3 year old grapevine cultivar Corvina in Italy, were able to
inhibit the growth of N. parvum in vitro. The B. methylotrophicus 3R1 strain expressed
the strongest antifungal activity [49]. Another bacterial species, Pseudomonas protegens
MP12, isolated from Italian forest soil, and the strain P. protegens DSM 19095T significantly
inhibited the mycelial growth of N. parvum when assessed in vitro [50]. Two strains of
P. agglomerans (S1 and S3) and one of Paenibacillus sp. (S19), isolated from grape berries
and wood tissue, respectively, inhibited the growth of N. parvum via the production of
phenylethyl alcohol, an antifungal volatile compound, while Paenibacillus sp. directly
inhibited N. parvum via antibiosis [22].
In a recent study, Bustamante et al. (2022) evaluated the antagonistic activity of
1344 endophytic and rhizospheric bacterial isolates against N. parvum. These bacterial
strains were isolated from different grapevine cultivars in California. The result showed that
172 isolates inhibited N. parvum growth by more than 40% in the dual-culture assay. These
bacteria belonged to the species B. velezensis (154 isolates), Pseudomonas spp. (12 isolates),
Serratia plymuthica (two isolates), and other genera (four isolates) [51]. In the same study,
it was reported that B. velezensis (two strains), Pseudomonas chlororaphis (two strains), and
Serratia plymuthica (two strains) reduced the mycelial growth of N. parvum via their agar-
diffusible metabolites. However, they gave a low inhibition on N. parvum mycelial growth
via the production of volatile organic compounds [51].
With regard to in planta assays, several bacteria were tested to fight N. parvum infection
on young plants, but no protection was observed with Acinetobacter radioresistens, B. firmus,
B. ginsengihumi, B. licheniformis, B. pumilus, Brevibacillus reuszeri, Curtobacterium sp., Enter-
obacter cowanii, Paenibacillus barengoltzii, Paenibacillus illinoisensis, Paenibacillus polymyxa,
Paenibacillus turicensis, and Xanthomonas sp. [1,52]. However, other bioassays were more
J. Fungi 2023, 9, 638 8 of 37

positive in terms of biocontrol protection, such as the one conducted under greenhouse
conditions by Haidar et al. (2021). They showed that Enterobacter sp. S24 and B. firmus
S41, isolated from grapevine wood, reduced the internal necrosis lesion length caused by
N. parvum. These authors demonstrated that, in addition to the in vitro inhibition of N.
parvum by P. agglomerans S1 and Paenibacillus sp. S19, these bacterial strains were able to
protect young grapevine from N. parvum infection via an indirect mechanism, i.e., induction
of plant resistance. They showed also that, among the various modes of application of these
potential biocontrol agents on plants, preventive inoculation on the stem was the most
efficient in controlling N. parvum [22]. Other studies showed that P. agglomerans reduced the
length of necrosis caused by N. parvum by 30% on grafted and by 32.3–43.5% on nongrafted
cutting stems of Cabernet Sauvignon cultivar under greenhouse conditions [52,53].

2.1.5. Biocontrol Using Actinobacteria

Regarding actinobacteria, 40 endophytic actinobacteria isolated from grapevine cv.
Sauvignon Blanc and identified as Streptomyces spp. were tested for their antagonistic
activity against N. parvum. Among them, 29 strains highly or moderately inhibited the
growth of this pathogen in vitro [54].
A wide variety of MBCAs have been tested in vitro and in planta against N. parvum with
Trichoderma spp. as the most tested biocontrol agent. The majority of Trichoderma strains
showed a very good efficiency in vitro; some of these strains showed a constant efficiency
in the greenhouse and the vineyard. Generally, a constant efficiency was observed for
other fungal isolates, such as C. rosea that gave a good antagonistic activity in vitro and in
planta under controlled conditions, and for F. proliferatum that inhibited the pathogen both
in planta and in the vineyard. The oomycete P. oligandrum also showed a good efficiency
under greenhouse conditions, but studies in vineyards are needed to confirm the efficiency
achieved in vitro and in greenhouses. Regarding bacterial strains, most of them were highly
effective in planta and/or in vitro. Strikingly, only one bacterial strain was tested in the
vineyard, but its effectiveness was inconsistent.

2.2. Biological Control of Diplodia seriata

Although less virulent than N. parvum, Diplodia seriata is one of the most aggressive
species isolated from diseased grapevines worldwide [19,21,55]. Various MBCAs have been
tested against this pathogen in vitro, in the nursery, and in the field to protect wounds. As
usual, depending on the strains, various levels of protection were obtained.

2.2.1. Biocontrol Using Trichoderma

In vitro Trichoderma spp. strains isolated from Southern Italy by Úrbez-Torres et al. (2020)
overgrew D. seriata mycelium. T. atroviride was the most efficient with a percentage radial
growth inhibition of 69.6%. According to Kovács et al. (2014), 10 Trichoderma spp. iso-
lated from the grapevine trunk also overgrew D. seriata mycelium in vitro [56]. T. atroviride
strains, USPP-T1 and USPP-T2, had an inhibitory effect on D. seriata, via the production
of secondary metabolites [34]. Indeed, an inhibitory zone between the colonies of these
two strains and that of D. seriata was observed, with hyphal disintegration of the pathogen.
Kovács et al. (2021) tested two Trichoderma strains identified as T. afroharzianum (strain TR04)
and T. simmonsii (strain TR05) isolated from grapevine cordon wood. These two strains
overgrew the pathogen colony, along with hyphal coiling and penetration in pathogen hy-
phae, suggesting mycoparasitism as the mechanism of action [31]. Recently it was reported
that 26 isolates of Trichoderma including species T. asperelloides, T. atroviride, T. harzianum,
T. koningii, T. tomentosum, T. canadense, and T. viticola significantly inhibited the growth of
D. seriata in vitro [37].
Regarding Trichoderma endophytes, Silva-Valderrama et al. (2021) reported that the
Trichoderma sp. strain Altair isolated from grapevine inhibited D. seriata growth as early as
7 days in vitro. Light microscope observations revealed that this Trichoderma strain produced
hyphal coils when it interacted with two D. seriata colonies, suggesting mycoparasitism
J. Fungi 2023, 9, 638 9 of 37

as a mode of action [30]. According to Wallis (2021), two other endophytes, T. atroviride
(ATCC 74058) and T. harzianum (ATCC 26799), were efficient in reducing D. seriata growth
by over 75%. The T. harzianum strain was qualitatively the most efficient in controlling
the pathogen as it outcompeted D. seriata for carbon and nitrogen sources [35]. In the
literature, strains belonging to the Trichoderma genus, i.e., Trichoderma sp., T. longibrachiatum,
T. harzianum, T. atroviride, T. afroharzianum, and T. simmonsii, were highly efficient in com-
peting in vitro against D. seriata, but this was also observed with strains from other genera
such as C. rosea, F. proliferatum, and Cladosporium sp. [1,30,31].
In planta experiments were usually conducted with Trichoderma spp. to protect prun-
ing wounds. For instance, T. paratroviride, Trichoderma sp., and two strains isolated from
P. persica, i.e., T. koningiopsis and T. guizhouense, controlled D. seriata infection by 89–94% on
pruning wounds when challenged with the pathogen at least 21 days after treatment [33].
Seven Trichoderma spp. isolates were tested on plated detached grapevine canes under con-
trolled greenhouse conditions to protect pruning wounds from D. seriata; all strains showed
moderate or high ability to protect pruning wounds from this pathogen. T. harzianum,
T. atroviride, and T. asperelloides were the most effective with a mean percentage disease
control of 97–100%, 21 days after treatment [37]. Kotze et al. (2011), in an experiment in
South African vineyards, interestingly observed that the T. atroviride strain USPP-T1 re-
duced the incidence of D. seriata by 85% after 8 months. In parallel, these authors evaluated
eight strains of three commercial products belonging to the species T. atroviride (AG3, AG5,
and AG8) and T. harzianum (AG2, AG11, Agss28, Biotricho, and Eco77). These strains,
except one, were able to overgrow D. seriata and operated through mycoparasitism. The
three bioproducts were able to reduce D. seriata incidence on pruning wounds under field
conditions [34].

2.2.2. Biocontrol Using Other Fungal Genera

Fungi from the Chaetomium, Cladosporium, Clonostachys, Fusarium, and Lecanicillium
genera have been tested against D. seriata, some of which are endophytes. Silva-Valderrama
et al. (2021) reported that three C. rosea strains completely overgrew D. seriata in vitro by
day 21, but they had various modes of action. The antagonistic activity of the strain C. rosea
CoS3/4.24, isolated from the grapevine rhizosphere, was associated with both a secreted
antibiotic compound and mycoparasitism. Indeed, the secreted metabolites of C. rosea
CoS3/4.24 reduced the growth of D. seriata by 47.2% and changed its colony morphology,
whereas hyphal coiling, associated with mycoparasitism, was observed in the confronting
zone of the two fungal cocultures. For the two other strains, the antagonistic activity of
C. rosea R36.1 was due only to mycoparasitism, while that of C. rosea CoR2.15 was due only
to antibiosis. In in planta trials, the strain CoS3/4.24, with two modes of action, was used,
and a significant growth inhibition of D. seriata was observed in all assays [30].
Other endophyte strains from various fungal genera have displayed antagonistic
activity in vitro against D. seriata. In dual culture, Cladosporium sp. acted on D. seriata
via antibiosis, whereby the growth inhibition (42.46%) of D. seriata operated through
metabolites secreted by the antagonist [30]. For Chaetomium sp., its mechanism of action
was related to a slow mycoparasitism, as its hyphae penetrated and coiled around those
of D. seriata on day 30 of coculture [30]. Wallis (2021) reported that the endophytic strain
of Lecanicillium lecanii (ATCC 46578) reduced D. seriata growth in vitro by about 20%, via
direct antagonism and competition for carbon and nitrogen sources [35]. In the experiment
of Blundell et al. (2021), two other A. pullulans strains (coded UCD 8189 and 8344), isolated
from grapevine sap and cane tissue from healthy Chenin Blanc cultivar, caused significant
inhibition of D. seriata radial mycelial growth, but no inhibitory effect was obtained in the
volatile assay.
In the study of Pinto et al. (2018), conducted in planta on grapevine cuttings cv.
Chardonnay, another fungal endophyte, A. pullulans strain Fito_F278, isolated from leaves
of V. vinifera in Portugal, was reported to have an indirect effect on D. seriata growth. This
strain promoted the induction of some plant defense responses in cutting plants, 1 week
J. Fungi 2023, 9, 638 10 of 37

after D. seriata inoculation. For instance, the expression of genes encoding plant defense
proteins, such as PR protein 6 (PR6) and -1,3-glucanase (Gluc), were upregulated [57]. In
addition to plant defense induction, these authors suggested that this A. pullulans Fito_F278
strain was also able to compete with GTD fungi in the field, as it colonized the grapevine at
an endophyte and epiphyte level. F. proliferatum was reported to be a pathogen for several
crops, but it had an antagonistic effect on the oomycete Plasmopara viticola, the causative
agent of grapevine downy mildew [58]. It limited the growth of D. seriata in vitro through
antibiosis and direct antagonism [32].

2.2.3. Biocontrol Using Bacteria

Regarding bacteria, experiments with strains from Bacillus and genera isolated from
grapevine organs have been conducted. A B. subtilis strain isolated from the arm’s wood
of the cultivar Chenin Blanc that expressed Eutypa symptoms was highly efficient in vitro
against D. seriata [34]. Its antagonistic activity was attributed to antibiotic compound
production and diffusion, causing hyphal malformation such as swelling [34]. In the field,
B. subtilis reduced the incidence of D. seriata in fresh pruning wounds of Chenin Blanc and
Merlot grapevine cultivars when the pathogen was inoculated 7 days after the biocontrol
treatment [34]. Blundell et al. (2021) isolated two B. velezensis strains from sap and cane
tissue of grapevine, which significantly inhibited D. seriata in dual culture. Bustamante
et al. (2022) showed that 172 endophytic and rhizospheric bacterial isolates, including B.
velezensis (154 isolates), Pseudomonas spp. (12 isolates), Serratia plymuthica (two isolates),
and four isolates from other genera, inhibited the growth of D. seriata by more than 40%
in vitro. However, when a bacterial strain of Burkholderia phytofirmans was used in an in
planta bioassay, it had no efficiency in inhibiting D. seriata infection [1]. To the best of our
knowledge, no actinobacteria or oomycete strains have been tested against D. seriata.
Overall, among the fungal strains, there was a high and consistent efficacy of Tricho-
derma isolates tested in vitro and in greenhouse/field conditions. As for bacteria, most
of them were highly efficient in vitro and in greenhouse conditions. However, only one
bacterial isolate was tested in planta, and its efficiency was not maintained over time.
Therefore, more studies are required to understand and evaluate the ability of bacteria in
more realistic conditions. To the best of our knowledge, no studies are available on the
antagonistic activity of oomycetes or actinobacteria against D. seriata.

2.3. Biological Control of Lasiodiplodia theobromae

Lasiodiplodia theobromae which is frequently found in tropical and subtropical regions,
is the most representative and aggressive species of the genus Lasiodiplodia involved in
grapevine Botryosphaeria dieback [59,60]. The taxonomy of Lasiodiplodia was recently
revised. As a consequence, fungal isolates previously reported as L. theobromae were reclas-
sified as new species. A number of species were then reduced to synonyms [61]. Potential
bacterial and fungal MBCAs were tested in vitro and in planta against this pathogen.

2.3.1. Biocontrol Using Fungi

Strains from various species of the genus Trichoderma have been assessed to con-
trol L. theobromae, such as T. atroviride, T. harzianum, T. koningiopsis, T. asperellum, and
T. asperelloides [62]. In vitro experiments carried out by Kotze et al. (2011) showed that
Trichoderma spp. strains had various modes of action. Indeed, one T. harzianum strain
(i.e., AG2) acted on L. theobromae via mycoparasitism, while another T. harzianum strain
(i.e., Biotricho) and two of T. atroviride (i.e., AG3 and AG5) likely had both antibiosis and
mycoparasitism as modes of action. However, in dual culture, the targeted fungus was
able to defend itself, as likely seen when T. atroviride strain AG8 and those of L. theobromae
inhibited each other [34]. In another experiment, strains of T. harzianum, T. asperelloides,
T. asperellum, and T. koningiopsis were substantial antagonists to L. theobromae 14 days after
dual inoculation [62]. In the same study, the strain T. asperelloides, coded 02/03, showed
endophytic penetration capacity in grapevine cane; in an in planta assay on healthy Ni-
J. Fungi 2023, 9, 638 11 of 37

agara Rosada grapevine shoots, this strain had a preventive and curative capability to
control L. theobromae, by protecting the pruning wounds from L. theobromae at 20 days post
inoculation [62].
In the field, the species T. atroviride was identified as a promising candidate to protect
pruning wounds against L. theobromae [1,34]. Kotze et al. (2011) showed that two T. atroviride
strains obtained from a vineyard in South Africa were effective enough to reduce the
incidence of L. theobromae by 92% when the pathogen was applied 7 days after biocontrol
treatment. Light microscope observation revealed a coiling between T. atroviride and
L. theobromae’s hyphae, suggesting mycoparasitism as the mechanism of action. Only
another endophyte fungal species, i.e., Epicoccum purpurascens, displayed efficiency in
controlling L. theobromae in vitro [1,63].

2.3.2. Biocontrol Using Bacteria

B. subtilis and Bacillus sp. (AG1) were the only bacterial antagonists to be tested against
L. theobromae [1,34,64]. In a dual-culture assay, B. subtilis inhibited L. theobromae growth,
and an inhibition zone was observed, associated with swilling and malformation of the
pathogen’s hyphae. Kotze et al. (2011) suggested that this effect could be attributed to
antibiotic substance production. For a B. subtilis (AG1) isolated from grape wood tissues
affected by Esca (reclassified as B. amyloliquefaciens in 2012 [65]), Alfonzo et al. (2009)
showed that the metabolites produced were, in part, responsible for its inhibitory effect
against L. theobromae. Under field conditions, pruning wounds treated with B. subtilis
showed significantly lower L. theobromae incidence 8 months after its inoculation [34].
Overall, only Trichoderma strains and one isolate of Epicoccum purpuascens have been
studied in vitro to control L. theobromae. Among the Trichoderma isolates, some efficiently
controlled the pathogen in vitro and under field conditions. Only two bacterial strains
have been tested, and both of them were effective. One of them (B. subtilis) showed great
efficiency in vineyards to protect pruning wounds.

2.4. Biocontrol of Neofusicoccum australe and Other Botryosphaeria Dieback-Associated Fungi

Experiments with fungi and bacteria with potential biocontrol properties were car-
ried out to fight Neofusicoccum australe, one of the most virulent species associated with
botryosphaeria dieback [66].
To control N. australe, Kotze et al. (2011) used Trichoderma strains, from commercial
products, i.e., T. harzianum and T. atroviride, or isolated from grapevine in South Africa,
i.e., two T. atroviride (USPP-T1 and USPP-T2) strains, and one of B. subtilis. These strains
inhibited the pathogen in vitro, by stopping the pathogen growth when the two colonies
entered in contact (T. harzianum Eco 77) and/or by establishing an inhibiting zone be-
tween the colonies (T. harzianum ag 11). The pathogen growth was first stopped then
overgrown (USPP-T1 and USPP-T2) or immediately overgrown, as observed with the
strains T. harzianum AG2, AG11, AG28, and Biotricho, as well as T. atroviride AG3, AG5, and
AG8 [34]. Under field conditions, in order to protect pruning wounds from N. australe, the
T. atroviride strain coded USPP-T1 was the most efficient with 78% reduction in incidence
8 months after pathogen inoculation [34].
As for Botryosphaeria dothidea, Botryosphaeria stevenssi (currently named Diplodia
mutila), Diplodia corticola, Neofusicoccum luteum, Neofusicoccum mediterraneaum, and other
pathogens associated with Botryosphaeria dieback, T. atroviride controlled almost all pathogens
in vitro, while T. gamsii was effective against B. stevenssi (D. mutila) in in vitro studies [1].
T. atroviride controlled N. luteum and N. mediterraneaum efficiency in in planta experiments [1].

3. Biocontrol of Esca
Esca is a white rot disease caused by a set of fungal Ascomycetes and Basidiomycetes
on the wood of grapevines [1,2]. The colonization of grapevine trunk and cordon woody
tissues by fungi, mainly Phaeomoniella chlamydospora, Phaeoacremonium minimum, and Fomi-
tiporia mediterranea, causes various types of necrosis. Bruno et al. (2020) suggested that
J. Fungi 2023, 9, 638 12 of 37

these three fungi disturb various morphological, physiological, and biochemical functions
in grapevine during the vegetative period, subsequently affecting bleeding xylem sap and
leaves, flux, dynamic viscosity, and growth regulator activity. They also alter grapevine
phenol metabolism according to Bruez et al. (2021). In the literature, it is assumed that Esca
results from the successive and coordinated action of these pathogenic fungi; P. chlamy-
dospora reduces plant resistance due to its toxic activity, P. minimum affects cell wall integrity
through its enzymatic activity, and, at the last stage of the disease, F. mediterranea takes
advantage of the cellular degradations caused by the previous fungi to cause complete
degradation of wood tissues, resulting in white rot necrosis formation [1,67–70]. Recently,
Haidar et al. (2021) showed for the first time that the fungal ability to degrade wood was
strongly influenced by wood-inhabiting bacteria. They demonstrated that a cellulolytic and
xylanolytic Paenibacillus sp. strain displayed a synergistic interaction with F. mediterranea to
enhance wood degradation structures [71].

3.1. Biological Control of Phaeomoniella chlamydospora

Many MBCAs have been used to control P. chlamydospora, and antagonistic species
belonging to the Trichoderma genus have been tested as the most effective against this
pathogen [1]. For instance, T. atroviride, T. harzianum, T. hamatum, T. longibrachiatum, T. gamsii,
and Trichoderma sp., when tested in vitro against this pathogen and under greenhouse, field,
and nursery conditions, were effective in colonizing grapevine wounds or preventing and
reducing vascular streaking caused by P. chlamydospora [1,30,34].

3.1.1. Biocontrol Using Trichoderma

When hyphae of P. chlamydospora and those of Trichoderma species interacted, over-
growth of the pathogen, competition for nutrient, and direct antagonism were reported
as mechanisms in the literature. For instance, the endophyte strains of T. atroviride
(ATCC 74058) and T. harzianum (ATCC 26799) were able to outcompete or utilize more
carbon and nitrogen sources than P. chlamydospora, significantly reducing the growth of
the pathogen by 90% [35]. Kotze et al. (2011) also reported that commercial strains of
T. harzianum and T. atroviride, as well as two T. atroviride (USPP-T1 and USPP-T2) strains
isolated from the grapevine, overgrew P. chlamydospora after stopping its growth. The same
result was obtained by Silva-Valderrama et al. (2021), who reported that the endophytic
antagonist Trichoderma sp. Altair completely overgrew P. chlamydospora on day 21 of coincu-
bation in vitro. Recently, Spasova et al. (2022) created an ecofriendly hybrid nanomaterial
made of poly(l-lactic acid) fibers (PLLA) coated with chitosan and T. asperellum spores. Due
to its good mechanical properties, this nanomaterial ensured the viability of the T. asperellum
spores. When tested in vitro, it significantly suppressed the growth of P. chlamydospora [72].
Trichoderma spp. have been tested for their ability to protect grapevine pruning
wounds in experiments in nurseries and in the field. For instance, the isolate USPP-T1
reduced the incidence of P. chlamydospora by 77% 8 months after inoculation under field
conditions [34]. Mycoparasitism was suggested as the mechanism of action. It was reported
that the application of T. harzianum at rooting in an organic nursery reduced the rate of
P. chlamydospora infection over time [73]. Regarding vine cuttings and pruning wounds,
their protection against P. chlamydospora infection were evaluated in the nursery and in
the field [74]. Cuttings were dipped in a Trichoderma suspension of T. harzianum T39
(Trichodex® ) and T. longibrachiatum before or after callusing. In the case of pre-callusing
dips, conflicting results were yielded for the 3 years of the study; however, in the post-
callusing treatment, Trichoderma spp. led to a significant reduction in necrosis length, caused
by P. chlamydospora inoculated into the rootstock. As Trichoderma spp. and P. chlamydospora
were never in contact, Marco and coworkers (2004) suggested that an enhancement of the
grapevine defenses was responsible for the protective effect. In the same study, pruning
wounds were also protected against P. chlamydospora infection under field conditions, with
the two biocontrol agents T. harzianum T39 and T. longibrachiatum being reisolated 2 months
after spraying [74].
J. Fungi 2023, 9, 638 13 of 37

As for rootstock, soaking of the planting material naturally infected by P. chlamydospora

in Trichoderma formulation was applied in South African nurseries, reducing the incidence
of the pathogen in the rootstock cuttings [75]. Martínez-Diz et al. (2021) also dipped the
roots and the basal part of the plant in Trichoderma koningii TK7 suspension for 24 h before
planting, and the incidence of P. chlamydospora infection in the field on young grafted
Spanish Tempranillo cultivar was significantly reduced.

3.1.2. Biocontrol Using Other Fungal Genera

In addition to Trichoderma, other fungal genera have been used in the literature to
control P. chlamydospora, including endophyte isolates of Epicoccum spp. taken from the
woody tissue of the cultivar Touriga Nacional grown a vineyard in Portugal. E. layuense
E24 was the most efficient strain in vitro as it reduced P. chlamydospora growth by 79.9% [76].
Its mode of action was probably via competition for space as E. layuense E24 grew faster
than the pathogen, or via chemical interaction as it produced diffusible pigments on the
medium [76]. E. layuense E24 was, therefore, tested under greenhouse conditions on rooted
cuttings of two grapevine cultivars against P. chlamydospora. E. layuense E24 colonized the
wood without impairing plant growth or inducing the appearance of symptoms in leaves or
wood. It reduced the frequency of the pathogen re-isolation and the brown wood streaking
length in Cabernet Sauvignon and Touriga Nacional by 67.5% and 73.8%, respectively [76].
Other in vitro tests were conducted with C. rosea and Lecanicillium spp. strains. Rhizo-
spheric and endophytic strains of C. rosea were cocultured with P. chlamydospora in vitro,
and C. rosea almost completely overgrew (99.9%) P. chlamydospora by 21 days, presumably
inhibiting pathogen growth through antibiosis and mycoparasitism [30]. L. lecanii (ATCC
46578) reduced the growth of P. chlamydospora by 50% in vitro and was able to outcompete
it for carbon and nitrogen resources [35]. Five endophytic strains of C. rosea isolated from
grapevine cv. Cabernet Sauvignon were effective in inhibiting P. chlamydospora growth
in vitro; this inhibition was observed before C. rosea physical contact with the pathogen,
which led the authors to suggest that the pathogen growth inhibition was due to the
production of antibiotic compounds by C. rosea [40].
A strain (i.e., F2) of Fusarium oxysporum was isolated from a suppressive compost
amendment [77]. It reduced the growth of P. chlamydospora by 43% and its sporulation
by 90% in vitro at 28 days; nonetheless, no reduction in the discoloration length inside
the trunk was observed, even though the DNA amount of P. chlamydospora was reduced
by 82% in the presence of this antagonist [78]. The F2-treated grapevines also harbored
higher lignin levels. The F2 strain was re-isolated 90 days after treatment, suggesting that it
probably colonized the xylem tissues [78].
In planta under greenhouse conditions, the endophytic isolate C. rosea 19B/1 was
assessed against P. chlamydospora on 1 year old grapevine cuttings grown for 90 days in
greenhouse conditions in soil with 104 /g conidia of C. rosea 19/B1. The results showed
that the length of the necrotic lesions caused by P. chlamydospora significantly decreased in
the case of cuttings planted in C. rosea-amended soil [40]. In this case, when the pathogen
growth was inhibited without any direct contact with C. rosea, the authors suggested two
possible mechanisms of action: the first by inhibiting the pathogen growth by antibiotic
compounds secreted in the soil or in the vascular tissues at the base of the cuttings, and
then transported by the sap to the point of P. chlamydospora inoculation; the second by
triggering the plant defense mechanism [40].

3.1.3. Biocontrol Using Oomycetes

As for oomycetes, experiments were conducted using P. oligandrum. This oomycete
was reported to protect grapevine against Esca pathogens, by inducing the plant defense
of Cabernet Sauvignon cuttings in controlled greenhouse conditions [53,79,80]. Yacoub
et al. (2016) and Daraignes et al. (2018) observed that the application of this oomycete
at the root level reduced P. chlamydospora necroses in the stem. As there was no contact
between the two microorganisms, with P. oligandrum applied in the soil surrounding the
J. Fungi 2023, 9, 638 14 of 37

roots and P. chlamydospora present in the aerial parts, Yacoub et al. (2016) pointed out
an enhancement of plant defense responses subsequent to pathogen infection. Six genes
involved in various plant defense pathways, including PR proteins, phenylpropanoid
pathways, oxylipin, and oxidoreduction systems, were more significantly expressed in the
presence of the oomycete [80]. This P. oligandrum induced plant systemic resistance and
was associated with the promotion of jasmonic/ethylene signaling pathways [79]. The
effects of the combination of P. oligandrum with Pantoea agglomerans or Bacillus pumilus in
young grafted grapevines under greenhouse conditions against P. chlamydospora were not
significantly different from the single bacterial strain applications; hence, no synergic effect
between these MBCAs took place in protecting against this pathogen [53]. Under field
conditions, the strain P. oligandrum Po37 significantly reduced P. chlamydospora infection on
young grafted Spanish Tempranillo cultivar [81].

3.1.4. Biocontrol Using Bacteria

Although strains from the Bacillus genus have been extensively used, strains from
other genera, i.e., Acinetobacter, Brevibacillus, Curtobacterium, Enterobacter, Paenibacillus, and
Pantoea, have also been tested for their ability to control P. chlamydospora.
Bacteria have been isolated from grapevine and tested for their efficacy in controlling
P. chlamydospora. Some experiments showed that, when B. subtilis interacted directly with P.
chlamydospora hyphae, swelling and malformation on the pathogen’s hyphae were observed,
suggesting antibiosis as the most likely mechanism of action [34]. Alfonzo et al. (2009),
showed that heat-stable metabolites of B. subtilis AG1 inhibited P. chlamydospora growth [64].
In the vineyard, when B. subtilis was applied on the surface of fresh pruning wounds in a
South African vineyard, there was a decrease in P. chlamydospora incidence 8 months after
infection [34].
Andreolli et al. (2016) isolated endophytic bacteria from 3 and 15 year old grapevine
stems of V. vinifera cultivar Corvina. They reported that Bacillus sp. 3R1 and 3R4, which
clustered with the species B. methylotrophicus, had a promising antagonist effect on P.
chlamydospora in vitro [49], and they were able to colonize the xylem tissue of grapevine. The
endophytic bacterial strain AG1 of B. amyloliquefaciens [65] produced heat-stable metabolites
and inhibited mycelial growth P. chlamydospora in vitro [64].
Haidar et al. (2016) assessed the antagonist activity of 46 bacterial isolates obtained
from grapevine wood or grape berries, sampled from a Bordeaux vineyard (France). Eight
among the 46 significantly reduced the necrosis length produced by P. chlamydospora on
rooted cuttings of a Cabernet Sauvignon cultivar under greenhouse conditions. Bacillus
pumilus (S32) and Paenibacillus sp. (S19) were the most efficient ones [82]. These two bacterial
isolates exhibited a direct action on P. chlamydospora, by producing volatile compounds and
a diffusible antibiotic substance in vitro that inhibited the pathogen growth; moreover, when
inoculated alone, they induced the expression of defense-related genes on the grapevine 4
days after their application. This effect was only maintained in the leaves of plants treated
with B. pumilus (S32) and P. chlamydospora, 15 days after pathogen inoculation. Haidar et al.
(2016) suggested that B. pumilus (S32) induced systemic resistance in grapevine.
In addition to the Bacillus and Paenibacillus genera, other bacteria isolated from
grapevine have been tested against P. chlamydospora. The same research group of Haidar
et al. (2016) evaluated the biocontrol capacity of Enterobacter sp. (S24), Paenibacillus sp.
(S18), B. reuszeri (S28, S31, and S27), Bacillus sp. (S34), P. illinoisensis (S13), Pantoea agglom-
erans (S1 and S3), and Bacillus firmus (S41) isolated from a Bordeaux vineyard against P.
chlamydospora. The result of the bioassay under greenhouse conditions on rooted cuttings of
Cabernet Sauvignon cultivar (INRAE, Bordeaux, France) showed that all bacterial strains
significantly reduced the length of the internal necrosis after the artificial co-inoculation of
the stem cuttings by P. chlamydospora; the strains B. reuszeri (S27) and Enterobacter sp. (S24)
were less effective. In each case, Haidar et al. (2016) provided evidence that the application
method, i.e., co-inoculation was prevented in the wood, and soil inoculation did not affect
the efficiency of these potential MBCAs. Some authors reported the inefficacy in vitro of
J. Fungi 2023, 9, 638 15 of 37

bacterial strains, such as one of Acinetobacter radioresistens and one of Curtobacterium sp.,
against P. chlamydospora [1].
Not all bacteria tested were isolated from grapevine; bacterial strains from the Pseu-
domonas genus, i.e., Pseudomonas protegens strain MP12, obtained from a forest soil sam-
ple, and P. protegens strain DSM 19095T, significantly inhibited P. chlamydospora growth
in vitro [50]. A mixture of two strains, Pseudomonas fluorescens and Bacillus atrophaeus (Stilo
Cruzial® ), was tested under field conditions on 2 and 3 year old Tempranillo cultivar in
Spain, against P. chlamydospora Petri disease, by soaking the roots and the basal part of the
plant in the two bacterial suspensions for 24 h before planting. However, this process was
inefficient against P. chlamydospora [81].
In a recent study, Paenibacillus alvei K165, isolated from the root tips of tomato plants
grown in solarized soils, was tested for its ability to control P. chlamydospora on a growth
medium simulating the xylem environment [83]. In planta, Gkikas et al. (2021) showed
that the growth and the sporulation of P. chlamydospora were not inhibited by this strain;
however, when tested on potted grapevines of cultivar Soultanina, the strain K165 reduced
the endophytic DNA amount of P. chlamydospora by 90%, and the wood discoloration length
in K165-treated vines was significantly reduced [78].

3.1.5. Biocontrol Using Actinobacteria

Álvarez-Pérez et al. (2017), evaluated the effectiveness of two actinobacterial strains
isolated from the root system of a 1 year old grafted V. vinifera cultivar Tempranillo. They
were an endospheric strain and a rhizospheric strain, Streptomyces sp. E1 and Streptomyces
sp. R4, respectively. In three experimental open-root field nurseries of young grafted V.
vinifera cv. Tempranillo plants, there was a significant reduction in the infection rates at the
lower end of the rootstock by P. chlamydospora, in the context of Petri disease [84]. However,
Martínez-Diz et al. (2021) showed that the antagonistic effect of the strains Streptomyces
sp. E1 and R4 put together was very low against Petri disease [81]. This points out the
complexity and the variability of plant protection induced by MBCAs.
Overall, most Trichoderma isolates have been reported to be effective in vitro, as well
as under field conditions for some strains. Other fungal genera showed a good efficiency
in vitro and/or in planta. The ability of the oomycete P. oligandrum and some actinobacteria
isolates were also evaluated and gave promising results in the control of P. chlamydospora.
Indeed, the growth of this pathogen was inhibited by numerous bacterial strains in vitro, as
well as in planta under controlled conditions.

3.2. Biological Control of Phaeoacremonium minimum

To control the Esca-associated fungus P. minimum, several strains of fungi, bacteria,
oomycete, and actinobacteria have been used in various assays.

3.2.1. Biocontrol Using Fungi

Chaetomium spp., Epicoccum spp., Lecanicillium lecanii, and Trichoderma spp. were
among the fungal strains evaluated [1,35,76,85].
Strains of Trichoderma spp. have been used extensively against P. minimum. In the ex-
periment conducted by Kotze et al. (2011) in vitro, commercial T. atroviride and T. harzianum
strains, alongside two T. atroviride strains (coded USPP-T1 and USPP-T2), were able to stop
the growth of the pathogen, with some strains coiling or disintegrating the pathogen hy-
phae. The mode of action of Trichoderma spp. in stopping P. minimum has been extensively
studied using phenotype microarrays [35], suggesting that they may compete on “nitrogen
plus carbon” and “carbon” sources with this pathogen. Wallis (2021) proposed direct
antagonism and competition for nutriment as the main mechanism of action. Nanomaterial
made of poly(l-lactic acid) fibers (PLLA), in which T. asperellum spores were incorporated,
significantly inhibited the growth of P. minimum in vitro [72].
An experiment conducted under semi-field conditions on potted vine plants in a
protected environment also provided evidence of antifungal activity within the plants.
J. Fungi 2023, 9, 638 16 of 37

Carro-Huerga et al. (2020) showed that the endophyte strain Trichoderma T154 colonized
the xylem vessels, fibers, and parenchymatic tissues inside the wood up to 12 weeks after
inoculation. They also showed a reduction in plant colonization by P. minimum. These
authors observed that the antagonistic effect of this strain was related to mycoparasitism,
mainly via the adhesion of spores to the pathogen hyphae and competition for a niche
by colonizing the xylem vessels [86]. Under field conditions on young grafted grapevine
cultivar Tempranillo, the strains T. atroviride SC1 and T. koningii TK7 significantly reduced
P. minimum infection [81].
Some endophytic fungal strains have been used to protect grapevines against P. mini-
mum. Three strains (Epicoccum layuense, Epicoccum mezzettii E17, and Epicoccum layuense E33
isolated from the wood of grapevines (cv. Touriga Nacional, Portugal), were able to inhibit
P. minimum in vitro, while other strains isolated from the same vineyard were not effective
against this pathogen [38]. Due to the fast-growing ability of these antagonists, along
with the diffusion in the culture medium of pigments, Del Frari et al. (2019) suggested
that antagonism was primarily due to competition for space and nutrients, as well as
probably chemical interaction. Another fungus, Chaetomium sp., had a significant in vitro
efficacy against P. minimum [1]. Geiger et al. (2022) studied the antagonist capacity of five
endophytic strains of C. rosea isolated from the grapevine in vitro and demonstrated that
none of these strains were effective in inhibiting P. minimum growth.
Under greenhouse conditions, the strain E. layuense E24, isolated from cane woody
tissue, was tested in vivo on two young grapevine cultivars. The wood symptomatology
caused by P. minimum was significantly reduced when interacting with E. layuense E24, as
well as unevenly between cultivars, with the best reduction for Cabernet Sauvignon (82%)
compared to Touriga Nacional cultivar (31.3%) [38].

3.2.2. Biocontrol Using Oomycetes

A biocontrol oomycete, P. oligandrum (strain Po37), significantly reduced P. minimum
infection associated with Petri disease on grafted young grapevine cultivar Tempranillo
under field conditions in Spain [81].

3.2.3. Biocontrol Using Bacteria and Actinobacteria

Bacterial strains have been tested against P. minimum. A number of these strains
were isolated from grapevine. Among them, many belonged to the genus Bacillus. The
antagonistic activity of B. subtilis was assessed in vitro and under field conditions against
P. minimum. In a dual-culture assay, the growth of this pathogen was inhibited by the
antagonistic bacteria isolate, and antibiosis was hypothesized as the mechanism of action,
as suggested by the malformations and swelling seen on pathogen’s hyphae [34]. Two
endophytic strains of Bacillus sp. isolated in Italy from a 3 year old V. vinifera cultivar
Corvina caused significant in vitro inhibition of P. minimum growth [49]. Another endo-
phytic strain of Bacillus licheniformis was isolated from V. vinifera cv. Glera and inhibited the
growth of P. minimum in dual culture [87]. The antagonistic effect of the strain B. subtilis
AG1 isolated from grapevine wood tissues affected by Esca and its heat-stable metabolites
showed their efficacy against P. minimum [64]. In 2012 this strain was reclassified as B.
amyloliquefaciens [65].
Combinations of Bacillus strain with other bacteria have also been proposed. In a
recent study, Pseudomonas fluorescens plus Bacillus atrophaeus, mixed in the commercial
product Stilo Cruzial® , were assessed on young (2 and 3 years old) Spanish vineyard
cultivar Tempranillo. This biocontrol product was applied by soaking the root and the basal
part of the plant in a suspension of the two bacterial strains 24 h before planting; however,
no effect was observed against P. minimum [81].
Two strains of Pseudomonas proteges exhibited antagonistic activity in vitro against P.
minimum [50]. The growth of P. minimum in vitro was highly inhibited by S. plymuthica, B.
velezensis, and P. chlororaphis [51].
J. Fungi 2023, 9, 638 17 of 37

Other bacteria and actinobacteria were isolated from the wood tissue of symptomatic
and asymptomatic grapevine of two cultivars Glera grafted onto SO4 rootstock and Sylvoz-
trained 20 year old plants [88]. Among the 38 selected bacterial strains, 24 were clustered
with the Actinobacteria branch, in addition to 13 with Rhizobiales and one with Pseu-
domonadales. Most of these strains were able to overgrow P. minimum in vitro, and three of
them showed high biocontrol potential against this pathogen (one of the three strains was
identified as Micromonospora sp.).
Other actinobacteria strains showed good efficiency in reducing the infection rates at
the lower end of the rootstock on young grapevine in the field [84].
Overall, most Trichoderma spp. strains tested in vitro significantly reduced P. minimum
growth. Only a few strains were evaluated in planta, some of which showed good potential
in controlling this pathogen. Other fungal and bacterial genera showed promising results
in vitro and/or in planta with a constant efficiency, whether in vitro or in planta. Regarding
the oomycete P. oligandrum, only one study reported its ability to reduce grapevine infection
by P. minimum in the field.

3.3. Biological Control of Fomitiporia mediterranea

F. mediterranea is mainly isolated from sectorial and central white rot necrotic tis-
sues [89], and only a few experiments with MBCAs have been performed to control it.

3.3.1. Biocontrol Using Fungi

Among the fungi, Epicoccum strains isolated from cane woody tissue of cultivar Touriga
Nacional from a vineyard in Portugal were mainly used against F. mediterranea. E. mezzettii
E17 and E. layuense E7 strains inhibited the growth of F. mediterranea after 14 days of
growth on PDA medium [38]. E. mezzettii E17 overgrew this pathogen, suggesting that the
antagonistic activity is primarily due to competition for space and nutrients. On the other
hand, the strains assigned to E. layuense species inhibited F. mediterranea growth without
physical contact between the colonies and with pigments observed on the culture medium,
suggesting the production of chemical inhibiting compounds. The most efficient E. layuense
strain reduced the fungal colony size by 71.8%. Microscopic observation of the confronting
zone revealed that F. mediterranea responded by entangling their hyphae, forming hyphal
strands [38].

3.3.2. Biocontrol Using Bacteria

Several biocontrol bacteria; strains have been isolated from various wood tissues of
symptomatic V. vinifera (21 year old Sauvignon Blanc cultivar) from the Bordeaux region
(France). Haidar et al. (2021) tested the antagonistic activity of 59 of these bacterial strains
against F. mediterranea strain Ph CO 36, in vitro. Thirty-five strains out of 59 effectively
inhibited pathogen growth in a dual-culture assay, while 99% of them inhibited its growth
by secreting volatile compounds. The strains Pseudomonas sp. S45, Stenotrophomonas
sp. S180, and Novosphingobium sp. S112 were the most effective against F. mediterranea
in vitro (more than 50% inhibition in confrontation and volatile tests. Not all these bacteria
had a deleterious effect on the pathogen; an additional five strains, Enterobacter sp. S11,
Paenibacillus sp. S150, Weeksellaceae S259, Paenibacillus sp. S270, and Bacillus sp. S5, even
promoted the growth of F. mediterranea in dual culture [71].
Overall, the biocontrol of F. mediterranea started in 2019, and strains of the genera
Epicoccum have only been tested in vitro against this pathogen. Some of these strains
showed a high efficiency in controlling F. mediterranea in vitro. So far, there are no in planta
reports controlling this pathogen; thus, further studies aimed at selecting MBCAs against
this pathogen are required.

4. Biocontrol of Eutypa Dieback

Eutypa dieback is a severe disease of vineyards that has been known for over 60
years, mainly caused by the Ascomycete Eutypa lata [9,90]. However, reports showed that
J. Fungi 2023, 9, 638 18 of 37

some species of the family Diatrypaceae such as Eutypa leptoplaca, Cryptovalsa ampelina,
and Eutypella vitis could also be involved in Eutypa dieback [9,21,91]. Regarding E. lata,
ascospores are produced by perithecia on dead wood tissues after rain, before being released
and dispersed by the wind. Through fresh pruning wounds, these ascospores penetrate
and germinate in the xylem vessels, and the mycelium E. lata slowly colonizes the woody
tissues [92,93].
In Eutypa dieback, the mechanisms involved in foliar symptoms and wood necrosis de-
velopment are not well understood. Mahoney et al. (2003) reported that they may be caused
by several E. lata metabolites, such as eutypine, which is the most phytotoxic [9,94,95].
E. lata synthesizes eutypine in wood tissue, which is likely transported by the ascending
sap flow to the herbaceous parts of the vine, before penetrating the grapevine cells via
passive diffusion, and then accumulating in the cytoplasm due to ion-trapping mecha-
nism related to the ionization state of the molecule [96]. When this phytotoxin targets the
mitochondria, it causes inhibition and uncoupling of mitochondrial oxidative phosphoryla-
tion [9,96]. The enzymatic reduction of eutypine gives its corresponding alcohol, eutypinol,
which is not toxic to grapevine, suggesting this as a detoxification pathway for eutypine [9].
In addition to eutypine, other phytotoxic compounds are thought to be involved in foliar
symptoms [3,97,98]. For instance, Andolfi et al. (2011) reported that E. lata produced related
secondary metabolites, mainly acetylenic phenols, along with some low-molecular-weight
metabolites involved in the chelator-mediated Fenton (CMF) reactions that generate highly
damaging reactive hydroxyl radicals, likely inducing necrosis on grapevine wood tissue [3].

4.1. Biological Control of Eutypa lata

To control E. lata in vitro and in planta, very different results in terms of effectiveness
have been obtained with potential biocontrol fungal, bacterial, and actinobacterial strains.
The following species have been tested: Trichoderma atroviride, T. guizhouense, T. harzianum,
T. koningiopsis, T. longibrachiatum, T. paraviridescens, T. spirale, T. afroharzianum; T. simmonsii,
Lecanicillium lecanii, Fusarium lateritium, Rhodotorula rubra (yeast), Candida famata (yeast),
Penicillium sp., Alternaria alternata, and Cladosporium herbarum [31,33–35,42,99–101].

4.1.1. Biocontrol Using Trichoderma

In vitro, the mode of action of Trichoderma species against E. lata hyphae, which is
strain-dependent, has been extensively studied, involving either production of inhibit-
ing metabolites or mycoparasitism. For instance, John et al. (2004) showed that the
mycelial growth of E. lata was inhibited by volatile and nonvolatile metabolites produced
by three strains of T. harzianum (AG1, AG2, and AG3). Similarly, Kotze et al. (2011) ob-
served these two modes of actions with T. harzianum and T. atroviride strains obtained
from biocontrol commercials products, i.e., Biotricho® , Vinevax® , and Eco 77® , as well as
two T. atroviride strains (USPP-T1 and USPP-T2) isolated from South African grapevine.
At the microscopic level, the hyphae of T. atroviride AG5 and T. harzianum Eco 77 coiled
around those of the pathogen, indicating mycoparasitism activity, but there was also a clear
inhibition zone between the cultures of USPP-T1 and USPP-T2 strains and those of E. lata,
suggesting that antibiosis occurred, as shown by the production of volatile and nonvolatile
inhibiting metabolites. Kovács et al. (2021) reported that T. afroharzianum TR04 and T.
simmonsii TR05 strains, obtained from grapevine cordon wood, displayed mycoparasitism
against E. lata, as seen by the hyphal coiling and penetration of the pathogen’s hyphae.
Screening of strains from various Trichoderma species obtained from different ecosys-
tems, according to their antagonistic ability against E. lata, was conducted by Úrbez-Torres
et al. (2019). Regarding T. atroviride, the most efficient strain was the isolate T. atroviride
PARC1018 that inhibited the mycelial growth of this pathogen by 68.2%, while three other
strains from this species caused a slight inhibition (less than 50%) of E. lata. Regarding the
other Trichoderma species, T. harzianum PARC1019, Trichoderma sp. PARC1020, T. koningiopsis
PARC1024, and four strains of T. guizhouense, obtained from Italian P. persica, as well as
the strain T. paratroviride PARC1012, significantly reduced the E. lata mycelium growth by
J. Fungi 2023, 9, 638 19 of 37

more than 50%, whereas, for others strains, i.e., T. harzianum PARC1013, T. longibrachiatum,
T. paraviridescens, and T. spirale obtained from P. persica, this mycelial growth reduction was
lower [33]. E. lata growth was significantly reduced by about 70% by the endophytic strains
T. atroviride (ATCC 74058) and T. harzianum (ATCC 26799), which outcompeted or utilized
more carbon and nitrogen sources than E. lata, suggesting competition for nutrients as the
mechanism of action [35].
Trichoderma spp. were mainly implemented in field trials to protect grapevine pruning
wounds. In South Africa, Halleen et al. (2010) showed that, on Cabernet Sauvignon vine-
yards (8 and 10 years old) artificially infected by E. lata, protection of pruning wounds was
higher with chemical products (benomyl and flusilazole) than with Trichoderma biocontrol
products (Trichoseal-Spray, Eco 77® , and Biotricho® ). On four other South African young
vineyards (5–9 years old) naturally infected with E. lata, two of them with grape cultivars
Cabernet Sauvignon and Sauvignon Blanc, and two others with table grape cultivars, Red
Globe and Bonheur, Trichoderma-based biocontrol products, Vinevax® and Eco 77® , reduced
the natural infection of pruning wounds, but their efficiency varied according to the season
and the cultivars [101].
Another treatment consisted of brushing the wounds with spores of Trichoderma spp. [100].
Indeed, in a glasshouse assay, T. harzianum AG1 prepared in sterile distilled water or
three commercial formulations, i.e., Trichoseal, Trichoseal spray, and Vinevax, applied
by brushing within the first hour of pruning, reduced the recovery of E. lata when the
ascospores of the pathogen were inoculated 2 or 7 days after pruning [100]. The same
reduction in E. lata recovery was observed under field conditions in a South Australian
healthy vineyard (16 year old Cabernet Sauvignon cultivar) when spores of T. harzianum
or Vinevax were applied by brushing on fresh pruning wounds 1 or 14 days before the
inoculation of E. lata ascospores [100].

4.1.2. Biocontrol Using Other Fungi

In vitro, according to Wallis (2021), L. lecanii (ATCC 46578) did not significantly re-
duce E. lata growth, but outcompeted or utilized more carbon and nitrogen sources than
the pathogen. Blundell et al. (2021) investigated the biocontrol ability of two strains of
A. pullulans isolated from cane tissue and the sap of Chenin Blanc cultivar grapevines in
California. These two strains, UCD 8344 and UCD 8189, were inefficient in a dual-culture
assay against E. lata. Five endophytic C. rosea strains obtained from grapevine were effective
in inhibiting E. lata growth in vitro, without any direct contact [40].
John et al. (2005) showed that the treatment of pruning wounds by spores of the
saprophyte Fusarium lateritium reduced the recovery of E. lata when the antagonist was
applied at least 1 day before the pathogen in a glasshouse assay, as well as on 16 year old
healthy vineyards of the cultivar Cabernet Sauvignon in South Australian.
Munkvold and Marios (1993) evaluated the ability of 348 fungal strains isolated from
grapevine pruning wounds of cultivar Chenin Blanc to inhibit E. lata in vitro on excised
grapevine stems. Among these isolates, 49% did not reduce E. lata infection on the non-
autoclaved wood, and only 1% completely reduced the infection of the wood stems by this
pathogen. These authors conducted two field bioassays on 21 year old Thompson seedless
grapevines and showed that the two strains of F. lateritium and Cladosporium herbarum
significantly reduced the infection of pruning wounds by E. lata, when applied using a
brush immediately after pruning. Depending on the bioassay, this reduction was equal
to or greater than with the fungicide treatment, i.e., with benomyl. Regarding the modes
of action of the two strains, F. lateritium certainly acted via antibiosis, as it produced a
diffusible metabolite that inhibited E. lata in vitro; regarding C. herbarum, as it has a high
rate of hydrophobic conidia sporulation, competition for space through the colonization of
pruning wounds by these conidia was probably its main antagonistic mechanism [102].
Under greenhouse conditions, Geiger et al. (2022) conducted an in planta study on 1
year old grapevine cuttings grown for 90 days in greenhouse in soil with 104 /g conidia
of C. rosea 19/B1. They showed that C. rosea 19/B1 significantly reduced the length of the
J. Fungi 2023, 9, 638 20 of 37

necrotic lesions caused by E. lata. Because C. rosea inhibited the pathogen growth without
any direct contact with it, Geiger et al. (2022) suggested that the mechanism of action of
C. rosea was antibiosis or induction of the plant defense mechanism.

4.1.3. Biocontrol Using Bacteria and Actinobacteria

Strains from the following bacterial species have displayed some potential to act as
biocontrol agents against E. lata,: Bacillus cereus, Bacillus megaterium, B. subtilis, Bacillus
thuringiensis, B. velezensis, Erwinia herbicola (syn Pantoea agglomerans), Micrococcus kristianae,
Pseudomonas sp., Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia plymuthica, and
Stenotrophomonas maltophilia [34,42,103,104].
Among the Bacillus species, B. subtilis has been extensively used in vitro and under
field conditions.
In vitro, B. subtilis strains were isolated from the wood of arms of Chenin Blanc
grapevine expressing Eutypa dieback symptoms or from compost soil. The strain coming
from grapevine inhibited in vitro the mycelial growth of E. lata by 91.4% and the ascospore
germination by 100% [104]. In dual culture, B. subtilis caused malformations [34,104] and
swelling in E. lata hyphae [34]. As Ferreira et al. (1991) identified at least two antibiotic
substances that were responsible for the inhibition of E. lata mycelial growth and ascospore
germination, antibiosis was suggested as the mode of action of this B. subtilis strain [34,104].
Schmidt et al. (2001) also hypothesized antibiosis as the mode of action when they used
the liquid cultures of two B. subtilis strains isolated from boiled compost soil. They caused
at least 50% suppression of E. lata mycelial growth over 2 weeks on autoclaved discs of
perennial grape wood cultivar Müller Thurgau (over 10 years old).
From 10 year old cultivar Chenin Blanc grapevines in California (USA), Blundell et al. (2021)
isolated one bacterial strain from the sap and another one from the cane pith of V. vinifera,
which were identified as closely related to B. velezensis. These two strains inhibited E. lata
mycelial growth in vitro in both dual-culture and volatile organic compound assays. In the
dual-culture assay, an inhibition zone was observed between E. lata and Bacillus isolate
UCD 8347, which also significantly inhibited the growth of E. lata in the volatile assay.
Hence, the authors suggested that an antibiotic substance was produced by this isolate.
Under field conditions in South Africa, a B. subtilis strain isolated from grapevine
wood was used to control E. lata infection on different cultivars of young and mature
vineyards [34,104]. On a 4 year old Riesling vineyard, after pruning of grapevines, B. subtilis
suspension and its antibiotic extract were sprayed directly on the pruning wound surface,
while E. lata was inoculated 4 h after [104]. Nine months later, it was shown that the
pathogenic infection was significantly suppressed by the bacterial suspension (100%).
However, the antibiotic substance was ineffective in protecting pruning wounds from
E. lata infection [104]. Kotze et al. (2011) showed that, when this bacterial strain was
sprayed on fresh pruning wounds of grapevines from 10 year old Merlot and 18 year old
Chenin Blanc vineyards, with E. lata was inoculated 7 days afterward, E. lata incidence
was lower after 8 months compared to the control. Nevertheless, in another experiment
realized in South Africa in 8 and 10 year old Cabernet Sauvignon vineyards, no efficiency
was observed in protecting pruning wounds when using a suspension of bacterial strains
against the artificial infection of E. lata 24 h after spraying [101]. These variations in
efficiency were probably due to the difference in grapevine ages and cultivars used in each
study. The spraying methods, i.e., covering or not the pruning wounds after treatment, and
the interval of time between B. subtilis and E. lata inoculations also presumably influence
the success or the failure of the biocontrol protection.
In addition to B. subtilis, other bacterial species/genera have been tested against
E. lata. Schmidt et al. (2001) reported that B. cereus, B. subtilis, B. thuringiensis, Pseudomonas
aeruginosa, eight strains of Pseudomonas fluorescens and one of Stenotrophomonas maltophilia,
and an unidentified isolate belonging to the Enterobacteriaceae family inhibited E. lata
growth in a coculture assay. However, none of these strains showed efficacy in controlling
this fungal pathogen on discs of grapevine wood. In the same assay, an Erwinia herbicola
J. Fungi 2023, 9, 638 21 of 37

strain (reclassified as P. agglomerans) isolated from the rhizosphere of a Gramineae species

in west Germany [105], displayed antifungal activity against E. lata in vitro, as well as on
wooden discs [103]. Its culture filtrate contained siderophores and antifungal molecules
that inhibited E. lata growth on the wood discs; therefore, Schmidt et al. (2001) suggested
that its mechanism of action was probably due to both antibiosis and a competitive effect
under iron-limiting conditions. The same authors tested the potential of 104 Actinomycetes
isolates in vitro against E. lata both in dual culture and on discs of grapevine wood assay.
Seventeen isolates identified as Streptomyces sp. inhibited E. lata growth; one of these
unidentified Actinomycetes isolates (A123) showed the highest degree of E. lata inhibition
on wood, ranging from 70% to 100% over a 4 week period. Eighty percent of the unidentified
Actinomycetes isolates inhibited E. lata in dual culture, but only 11% were efficient in the
antagonism assay carried out on grapevine wood discs [103]. Recently, it was reported
that 30 endophytic actinobacteria isolates obtained from grapevine were able to inhibit or
moderately highly the growth of E. lata in vitro [54].
Munkvold and Marios (1993) showed that 60% of the 391 bacteria isolated from
pruning wounds of the cultivar Chenin Blanc reduced the infection by E. lata on grapevine
autoclaved stems, and only 2% (20 strains) were able to inhibit this pathogen. However,
under field conditions, the strains B. megaterium, M. kristianae, and P. fluorescens were
inefficient in protecting pruning wounds from E. lata infection, and the colonization of
pruning wounds by the pathogen was even enhanced in the presence of a B. megaterium
strain. Munkvold and Marios (1993) suggested that the high dose of ascospore inoculum of
E. lata used for the field experiment reduced the efficacy of the strains tested [102].
Overall, to biocontrol E. lata, many strains of Trichoderma have been evaluated in vitro
and in planta over the previous years. Some of them gave very good results in vitro and
under field conditions, but the efficiency of MBCAs depends on factors such as the season
of application and the grapevine cultivars. This was observed with some Trichoderma-based
products evaluated in the vineyards against E. lata. Other fungal strains such as C. rosea,
Fusarium lateritium, and L. lecanii effectively controlled E. lata in vitro and/or in planta.
Bacterial isolates were also evaluated, but they showed variable agreement between the
in vitro results and the results obtained in the field.

5. Biological Control with Currently Commercialized Products

As reported above, the grapevine’s defense responses are sometimes not sufficient
to cope with the development of GTD fungal pathogens. However, no highly efficient
treatment currently exists to prevent, protect, or even limit the progression of these diseases.
Only a few products are registered to reduce Esca foliar symptoms: a product based on a
foliar fertilizer mixture of calcium, magnesium, and seaweed (Algescar® , Natural Develop-
ment Group, Castelmaggiore, Bologne, Italy) [2,19,106,107], and a few Trichoderma-based
products registered in some countries. Furthermore, no commercial bacterial biocontrol
products are registered against GTD pathogens [52]. In this section, we review products
based on Trichoderma spp. strains to biocontrol GTD fungi.
Vintec® is a fungicide based on 2 ⇥ 1010 CFU/g of Trichoderma atroviride strain SC1
spores [55], in the form of dispersible granules, which is applied by spraying. The strain
T. atroviride SC1 is approved for use under EC 1107/2009 in several European countries as
a fungicide. It is widely used to control various fungal pathogens involved in grapevine
wood diseases such as P. chlamydospora, P. minimum, D. seriata, Botryosphaeria ribis, E. lata,
and Eutypa armenicae, as well as for crop protection against gray mold (Table 1). T. atroviride
SC1 was isolated from decayed hazelnut wood and selected as an MBCA for its high
colonization ability and its good lignocellulolytic capacity. In addition, this strain can
use mannose and galactose as carbon sources, which are the main components of the
hemicellulose of softwood [108]. T. atroviride SC1 has antagonistic activity against several
plant pathogens [109]; it is a fast-growing fungus that has no negative effects on plants but
enhances plant growth by promoting nutrient assimilation, in addition to quickly colonizing
the dead wood [110]. In nurseries, T. atroviride SC1 was more efficient when applied at
J. Fungi 2023, 9, 638 22 of 37

hydration stages to control P. minimum and P. chlamydospora infections [111]. According

to the producer’s recommendations, Vintec® should be applied when the environmental
temperature is equal to or higher than 10 C for a minimum of 5 h the day of the field
application [55]. Recently, Martínez-Diz et al. (2021) reported that soaking the roots and
the basal part of the V. vinifera Tempranillo cultivar in T. atroviride SC1 suspension for
24 h before planting reduced the incidence of certain GTD fungi [81]. T. atroviride SC1
was very effective in preventing P. minimum and P. chlamydospora infection on grapevine
pruning wounds in the field, as well as during the grafting process in nursery [111,112].
Berbegal et al. (2020) showed that this strain could reduce infections caused by some GTD
pathogens when new vineyards were planted.

Table 1. MBCAs officially registered for the management of GTDs in different countries through-
out the world. BPDB: Bio-Pesticides Database, accessible at http://sitem.herts.ac.uk/aeru/bpdb/
index.htm (accessed on 24 May 2023); AU: Australia; BE: Belgium; CA: Canada; CY: Cyprus;
CZ: Czech Republic; DE: Germany; EL: Greece; ES: Spain; EU: European Union; FR: France;
HR: Croatia; HU: Hungary; IT; Italy; KE; Kenya; LU: Luxembourg; MA: Morocco; NL: The Nether-
lands; NZ: New Zealand; PL: Poland; PT: Portugal; RO: Romania; SA: South Africa; SI: Slovenia;
TR: Turkey; UK; United Kingdom; USA: United States of America; VT: Vietnam; ZM: Zambia.

Target
Trade Name MBCAs Mode of Action Country References
Pathogen(s)/Disease
BE, CY, CZ,
Antibiosis; nutrient
P. chlamydospora, DE, EL, ES,
and space
P. minimum, D. seriata, FR, HR, HU, BPDB
Vintec® /Treadani1 T. atroviride SC1 competition;
E. lata, E. armenicae, IT, LU, NL, [113,114]
stimulation of plant
B. ribis, and grey mold PL, PT, RO, SI,
defenses
UK, NZ, USA

Fungus Esca (Phaeomoniella,

Phaeoacremonium) and
Competition for
botryosphaeria CY, ES, FR, IT,
Esquive® /Tri- T. atroviride space and
dieback, E. lata and PT, AU, NZ, BPDB [115]
Wall I-1237 nutriments;
also used for the SA, VT
mycoparasitism
control of root diseases
and damping-off
T. harzianum Competition for
Eco 77 Eutypa and botrytis SA; KE, ZM BPDB [115]
strain B77 space and nutrients
Antibiosis; Fungus involved in
Blindar/Cassat mycoparasitism; Esca, Botryosphaeria, USA, CA, EU
T. asperellum
WP/Remedier® / colonization of and Eutypa dieback Members
ICC012 & BPDB [115]
Escalator pruning wounds; Grapevine trunk ES, FR, IT, SI),
T. gamsii ICC080
Bioten WP nutrient and space disease; soil-borne MA, SI, TR
competition pathogens
Eutypa dieback (E. lata)
Mix Stimulation of the
Vinevax 5 strains of and botryosphaeria
systemic protective NZ, AU [115]
Bio-dowel T. atroviride dieback (Botryosphaeria
response
stevensii)
Eutypa dieback (E. lata)
black dead arm
5 strains of Competition for
Vinevax™ (Botryosphaeria spp.) NZ, AU [115]
T. atroviride space and nutrients
and Petri disease
(P. chlamydospora).

Chervin et al. (2022) determined that Vintec® significantly reduced the wood colo-
nization by P. chlamydospora and P. minimum on 1 year old canes of V. vinifera cv. Cabernet
Sauvignon, planted in pots. By conducting metabolomic studies, these authors showed
that the application of Vintec® alone induced a weak metabolomic response that was not
J. Fungi 2023, 9, 638 23 of 37

sufficient to stimulate plant defense mechanisms. Nevertheless, the application of Vintec®

with the pathogens attenuated the virulence, since some P. minimum and P. chlamydospora
metabolites were highly produced in the control condition but less produced in the pres-
ence of Vintec® [113]. This product also had an effect on the plant by priming its defense
mechanisms. It seems that Vintec® increased plant response with a stimulation of the
phenylpropanoid pathway with increasing amounts of stilbenoid pterostilbene, as well as
an increase in flavonoids. This allowed the authors to suggest a mechanism of action based
on competition and the stimulation of plant defense mechanisms [113]. By performing a
transcriptomic analysis, Romeo-Oliván et al. (2022) revealed that T. atroviride SC1 (Vintec® )
enhanced modifications in the gene response to GTD, both alone and during P. minimum
and P. chlamydospora infection. During infection by these pathogens, Vintec® promoted the
expression of genes related to the biosynthesis of stilbenes, phenols, and flavonoids, which
are metabolites known for their antifungal properties. It also modulated the expression of
some genes involved in hormonal signaling, especially those involved in auxin signaling.
Accordingly, the authors suggested that Vintec® enhanced the primary defense response of
the plant against Esca-associated pathogens [114].
Esquive® is another biological control product containing spores of the species T. atroviride;
the strain coded I-1237 was originally isolated from the soil (BPDB). This product is ap-
proved for use under EC 1107/2009 in Cyprus, France, Italy, Spain, and Portugal (BPDB), as
well as in Australia New Zealand, South Africa, and Vietnam [115], to prevent the infection
of grapevine pruning wounds by Esca and Botryosphaeria-associated pathogens, as well
as E. lata, and for the control of root diseases and damping off in fruits and vegetables
(Table 1). Esquive® contains 108 UFC/g of live T. atroviride I-1237 spore [55], in the form of
wettable powder used on leaves or via brush application on pruning wounds. The strain
I-1237 colonizes the wood after its penetration through pruning wounds and protects the
grapevine from GTD pathogens via various mechanisms of action, including the inhibition
of pathogenic fungal growth by competing for nutrients and mycoparasitism (Table 1).
Mounier et al. (2016) showed that the application of Esquive® on pruning wounds of ma-
ture grapevines for at least 2 years reduced plant mortality and leaf symptoms associated
with Botryosphaeria dieback, Eutypa dieback, and Esca [116]. Martínez-Diz et al. (2021)
evaluated the efficiency of Vintec® and Esquive® after their application on two mature
Spanish commercial vineyards (37 and 29 years old) during two seasons (2018–2019 and
2019–2020), but their results showed a low efficacy of these products against P. chlamydospora
and D. seriata in the two vineyards over 2 years [55].
Eco77 is a bioproduct approved for use to control Botrytis in zucchini, tomato, and
roses, as well as Eutypa dieback in grape in South Africa, Kenya, and Zambia [115]. This
product is available in the form of wettable powder that contains 2 ⇥ 109 spores/g of
T. harzianum B77, applied by spraying on pruning wounds. The protection conferred by
this strain is due to its ability to colonize the pruning wound and to compete for space and
nutrients, thus preventing E. lata infection. Kotze et al. (2011) found that this strain might
also produce antifungal metabolites in vitro, suggesting antibiosis.
Blindar is a mixture of two Trichoderma spp. strains, i.e., Trichoderma asperellum ICC012
and Trichoderma gamsii ICC080, that was approved for use on grapevine in the form of
wettable powder. This product contains 20 g/kg of each Trichoderma spp. Blindar protects
pruning wounds through various mechanisms of action: colonization of the wounds with
activity on GTD fungi via antibiosis and mycoparasitism, as well as growth inhibition
by competing for nutrients in the invasion sites (see BPDB website for more details).
It is applied on the grapevine by spraying at the beginning of the season because the
germination and growth of Trichoderma spores require favorable temperatures, and because
the grapevine bleeding sap is rich in sugars that favor spore development. The two
Trichoderma strains act via mycoparasitism (BPDB): T. asperellum on P. chlamydospora at 15 C
and T. gamsii at 10 C. These Blindar strains are available in other countries within other
commercial formulations, named Cassat WP, Escalator, Bioten WP, or Remedier® (Table 1).
Remedier® is, for instance, commercialized and used in Italy to reduce the incidence
J. Fungi 2023, 9, 638 24 of 37

of Esca and grapevine mortality in affected vineyards by protecting wounds from new
infections. Like Blindar, the Remedier® product contains 20 g/kg of T. asperellum ICC012
and 20 g/kg of T. gamsii ICC080 [13,117]. After multiyear treatment, this product provided
good results starting from the second or third year of application [1]. It was reported that
the spraying of solutions containing this product for 7 years at the phenological stage
of bleeding in three Italian vineyards reduced Esca symptoms by 22% [118]. Recently,
Di Marco et al. (2022) demonstrated the effectiveness of the preventive application of these
products under field conditions, early after pruning and yearly after planting [119].
Vinevax is another product that contains five strains of T. atroviride and is available in
two forms, Vinevax Biodowel and Vinevax™ Pruning Wound Dressing (Table 1), which are
approved and commercialized in Australia and New Zealand. The first one is in the form
of slow-release wood dowels, applied directly in the trunk to prevent and treat Eutypa
dieback (E. lata) and Botryosphaeria dieback (Botryosphaeria stevenssi syn. Diplodia mutila) by
stimulating the systemic protective response of the plant. Vinevax is also used on orchard
and ornamental trees [115]. The second one is a wettable powder applied by spraying
on grapevine pruning wounds. Its protective effect against airborne Eutypa ascospores is
due to its ability to durably colonize the pruning wounds. It is also used on orchard trees
against wood decay [115].

6. Mechanisms of Action of MBCAs against GTDs

The lack of effective strategies to manage GTDs and the need for the ongoing develop-
ment of biocontrol products have prompted scientists to evaluate the biocontrol potential
of numerous microbial strains against GTD fungi. After the ban of sodium arsenite, fungi
were used in initial studies on GTD pathogen biocontrol, and some Trichoderma strains
were registered and used in viticulture, but these products are not intended to specifically
manage GTDs. The two species T. atroviride and T. harzianum are frequently used to control
at least one of the GTD pathogens, and they are known to act via several mechanisms
of action, such as mycoparasitism and competition for space and/or nutrients (Table 2).
Within species of the Trichoderma genus (Table 2), the latter mode of action, i.e., competition
for space and/or nutrients, presumably plays an important role in controlling GTD fungi
since most pathogens penetrate grapevines through pruning wounds [33].
In comparison to fungal strains, the number of bacterial strains tested is high, with
strains belonging to Pseudomonas and Bacillus genus being the most tested against GTD
fungi; however, no bacterial products are currently available on the market. The mechanism
of action of these potential biocontrol bacteria has been less addressed in the scientific
literature, but antibiosis and induction of grapevine resistance by priming the expres-
sion of defense-related genes (Table 2) are the two most commonly cited. Among all
potential MBCAs, Actinobacteria are less studied for their antagonistic activity against the
GTD pathogens.
The oomycete P. oligandrum naturally colonizes the grapevine root system and protects
it via the induction of systemic acquired resistance against several GTD pathogens (Table 2).
Antibiosis, competition for nutrients and space, the production of siderophores and
hydrolytic enzymes, parasitism, and the induction of systemic resistance are the mechanism
of action exhibited by the various MBCAs assessed against GTD-associated fungi (Figure 1).
J. Fungi 2023, 9, 638 25 of 37
Table 2. The mechanism of action of the MBCAs against GTD-associated fungi.
MBCAs Strains Mechanisms of Action Targeted Pathogens References
Fungi
T. afroharzianum Mycoparasitism N. parvum, D. seriata, and E. lata [31]
T. asperelloides Competition for space L. theobromae, N. parvum, and D. seriata, [37,40,62]
T. asperellum Competition for nutrients and/or space P. chlamydospora, P. minimum, L. theobromae, and E. lata BPDB, [42,62,72]
Competition for space and nutrients, production P. chlamydospora, P. minimum, D. seriata, Botryosphaeria
BPDB,
T. atroviride of lytic enzymes, antibiosis, mycoparasitism, and ribis E. lata, N. parvum, N. australe, E. armenicae,
[1,33–35,37,62,108,113,115]
stimulation of plant defense mechanisms P. viticola, and N. mediterraneaum
T. canadense NA N. parvum and D. seriata [37]
T. gamsii Antibiosis and mycoparasitism P. chlamydospora and B. stevenssi BPDB, [1]
T. guizhouense Competition for nutrients and space N. parvum, D. seriata, and E. lata [33]
T. hamatum Competition for space and nutrients N. parvum, P. chlamydospora, and E. lata. [42]
Competition for space and nutrients,
Trichoderma P. chlamydospora, N. parvum, D. seriata, E. lata, P. viticola,
T. harzianum mycoparasitism antibiosis, and enhancement of [1,33–35,37,56,62,73]
P. minimum, N. australe, and L. theobromae
the grapevine defense response
T. koningii NA P. chlamydospora, P. mínimum, N. parvum, and D. seriata [37,81]
T. koningiopsis Competition for nutrients and space N. parvum, D. seriata, E. lata, and L. theobromae [33,62]
Competition for nutrients and space, and
T. longibrachiatum D. seriata, N. parvum, E. lata, and P. chlamydospora [33,56,73]
enhancement of grapevine defense response
T. paratroviride Competition for nutrients and space N. parvum, D. seriata, and E. lata [33]
T. paraviridescens Competition for nutrients and space N. parvum, D. seriata, and E. lata [33]
T. simmonsii Mycoparasitism N. parvum, D. seriata, and E. lata [31]
T. spirale Competition for nutrients and space N. parvum, D. seriata, and E. lata [33]
T. tomentosum NA N. parvum and D. seriata [37]
T. viticola NA N. parvum and D. seriata [37]
Competition for nutrients and space, and
Trichoderma sp. D. seriata, P. chlamydospora, P. minimum, and E. lata [30,33,56,86]
mycoparasitism
J. Fungi 2023, 9, 638 26 of 37
Table 2. Cont.
MBCAs Strains Mechanisms of Action Targeted Pathogens References
Production of diffusible metabolites in vitro and
E. layuense P. chlamydospora, P. minimum, and F. mediterranea [38]
competition for space and nutrients
Epicoccum Production of diffusible metabolites in vitro and
E. mezzettii P. chlamydospora, P. minimum, and F. mediterranea [38]
competition for space and nutrients
E. purpurascens NA L. theobromae [63]
F. lateritium Antibiosis E. lata [100,102]
F. oxysporum Colonization of xylem tissue (competition) P. chlamydospora [78]
Fusarium
Direct antagonism (antibiosis) and priming plant
F. proliferatum N. parvum and D. seriata [32]
defense response
The colonization of pruning wounds by its
C. herbarum E. lata, [102]
hydrophobic conidia (completion)
Cladosporium
Antibiosis and high rate of sporulation
Cladosporium sp. N. parvum, D. seriata, and P. chlamydospora [30]
(competition)
Aureobasidium A. pullulans Direct antagonism (stopped growth) N. parvum, D. seriata, and E. lata [42,102]
Candida C. famata NA E. lata [102]
Chaetomium Chaetomium sp. Mycoparasitism N. parvum, D. seriata, and P. chlamydospora [30]
D. seriata, N. parvum, P. chlamydospora, P. mínimum, and
Clonostachys C. rosea Antibiosis and mycoparasitism [30,40]
E. lata
N. parvum, D. seriata, P. chlamydospora, P. minimum, and
Lecanicillium L. lecanii Competition for space and nutrients [35]
E. lata
Penicillium Penicillium sp. NA E. lata [102]
Direct antagonism (secreted secondary
Purpureocillium P. lilacinum N. parvum, D. seriata, and P. chlamydospora [30]
metabolites)
Rhodotorula R. rubra NA E. lata [102]
Bacteria
Achromobacter Achromobacter sp. NA F. mediterranea [71]
J. Fungi 2023, 9, 638 27 of 37
Table 2. Cont.
MBCAs Strains Mechanisms of Action Targeted Pathogens References
B. amyloliquefaciens Antibiosis L. theobromae, P. chlamydospora, and P. minimum [65]
B. cereus Direct antagonism E. lata [103]
B. firmus NA N. parvum and P. chlamydospora [81]
B. licheniformis Direct antagonism P. minimum [89]
B. methylotrophicus Direct antagonism N. parvum, P. chlamydospora, and P. minimum [49]
Bacillus Induction of the expression of defense-related
B. pumilus N. parvum and P. chlamydospora [52,53,82]
genes
Antibiosis and induction of the expression of N. parvum, D. seriata, L. theobromae, N. australe,
B. subtilis [28,52,82,101,103,104]
defense-related genes P. chlamydospora, P. minimum, and E. lata
B. thuringiensis Antibiosis and competition for nutrient E. lata [103]
N. parvum, D. seriata, L. theobromae, P. minimum, and
B. velezensis NA [42,51]
E. lata
Bacillus sp. NA P. chlamydospora and F. mediterranea [52,71]
B. reuszeri NA P. chlamydospora [82]
Brevibacillus
Brevibacillus sp. NA N. parvum [49]
Brevundimonas sp. Brevundimonas sp. NA F. mediterranea [71]
Burkholderia Burkholderia sp. NA F. mediterranea [71]
Cedecea sp. Cedecea sp. NA F. mediterranea [71]
Chryseobacterium Chryseobacterium sp. NA F. mediterranea [71]
Curtobacterium Curtobacterium sp. NA F. mediterranea [71]
Enterobacter Enterobacter sp. NA N. parvum, P. chlamydospora, and F. mediterranea [71,82]
Frigoribacterium Frigoribacterium sp. NA F. mediterranea [71]
Erwinia Erwinia sp. NA F. mediterranea [71]
Herbiconiux Herbiconiux sp. NA F. mediterranea [71]
Kocuria Kocuria sp. NA F. mediterranea [71]
Luteimonas Luteimonas sp. NA F. mediterranea [71]
Lysinibacillus Lysinibacillus sp. NA F. mediterranea [71]
J. Fungi 2023, 9, 638 28 of 37
Table 2. Cont.
MBCAs Strains Mechanisms of Action Targeted Pathogens References
Microbacterium Microbacterium sp. NA F. mediterranea [71]
Novosphingobium Novosphingobium sp. NA F. mediterranea [71]
Olivibacter Olivibacter sp. NA F. mediterranea [71]
P. alvei NA P. chlamydospora [78]
P. illinoisensis NA P. chlamydospora [82]
Paenibacillus
Induction of the expression of defense-related
Paenibacillus sp. N. parvum and P. chlamydospora [22,52,82]
genes and antibiosis
P. protegens NA N. parvum, P. minimum, and P. chlamydospora [50]
P. fluorescens NA E. lata [102,103]
Pseudomonas P. chlororaphis NA N. parvum, L. theobromae, P. minimum, and E. lata [51]
P. aeruginosa NA E. lata [103]
Pseudomonas sp. NA F. mediterranea, N. parvum, and D. seriata [51,71]
Induction of the expression of defense-related
Pantoea P. agglomerans N. parvum, P. chlamydospora, E. lata, and F. mediterranea [52,53,71,82,103]
genes, antibiosis, and production of siderophores
Pedobacter Pedobacter sp. NA F. mediterranea [71]
Pigmentifaga Pigmentifaga sp. NA F. mediterranea [71]
Pseudoxanthomonas Pseudoxanthomonas sp. NA F. mediterranea [71]
Rahnella Rahnella sp. NA F. mediterranea [71]
Rhizobiaceae / NA F. mediterranea [71]
E. lata, N. parvum, D. seriata, L. theobromae, and
Serratia S. plymuthica NA [51,103]
P. mínimum
Sphingomonas Sphingomonas sp. NA F. mediterranea [71]
S. maltophilia NA E. lata [103]
Stenotrophomonas
Stenotrophomonas sp. NA F. mediterranea [71]
Variovorax sp. Variovorax sp. NA F. mediterranea [71]
Xanthomonaceae / NA F. mediterranea [71]
Actinobacteria
J. Fungi 2023, 9, 638 29 of 37
Table 2. Cont.
MBCAs Strains Mechanisms of Action Targeted Pathogens References
Streptomyces Streptomyces sp. NA P. chlamydospora, P. minimum, N. parvum, and E. lata [54,81,103]
Oomycete
Pythium P. oligandrum Induction of systemic resistance N. parvum and P. chlamydospora, [43,53,55,79,80]
NA: not available.
J. Fungi 2023, 9, 638 30 of 37

7. Factors Influencing Control Efficiency of the MBCAs

Variability and environmental factors, e.g., climate and soil type, have an influence on
the efficacy of the biological control agents in the field [120]. In the case of GTD biocontrol,
efficiency depends on the strains, as those belonging to the same species have various levels
of efficiency toward the same pathogen. For instance, various strains of T. harzianum had
different levels of efficiency against E. lata in vitro [33,34,99] and in planta [34,99]. The same
observation was made by Silva-Valderrama et al. (2021) for various strains of the species
C. rosea against N. parvum.
The same result was obtained with bacteria, whereby strains belonging to the same
species displayed different levels of efficiency when assessed against the same pathogen.
This was reported by Haidar et al. (2016) who conducted assays in vivo and observed
clear differences in the biocontrol efficiency of bacterial strains belonging to the same
species against N. parvum and P. chlamydospora. This dissimilarity also depends on the
pathogens targeted: (i) at the species level, Úrbez-Torres et al. (2020) showed that the
same strains of Trichoderma spp. had different behaviors against N. parvum, D. seriata,
and E. lata, while the same observation was reported by Kotze et al. (2011) when a
strain of Trichoderma spp. responded differently to interactions with N. parvum, D. seriata,
L. theobromae, P. chlamydospora, P. minimum, N. australe, E. lata, and P. viticola; (ii) at the
strain level, Mondello et al. (2019) showed that F. proliferatum was highly effective in vitro
against the strain N. parvum “Sainte Victoire”, but less effective against two other strains of
N. parvum tested under the same conditions.
This efficiency dependence on the pathogen/antagonist strains was also reported by
John et al. (2004) when metabolite production and action were considered. For instance,
T. harzianum AG2 volatile metabolites were the most effective in reducing the growth of
E. lata 280, while those of T. harzianum AG3 strongly inhibited E. lata CS-Ba.1 [99]. Because
the production of metabolites is strain-dependent, even within the same species [99], the
difference in efficiency depends strongly on the mechanism of action of MBCAs and the re-
sponse of the pathogens. For instance, in the study of Kotze et al. (2011), an inhibitory effect
between T. atroviride AG8 and L. theobromae in a dual culture was observed. This assumption
was supported by Kotze et al. (2011), who observed that some strains of T. atroviride dis-
played antibiosis against D. seriata, while other strains employed mycoparasitism toward
the same strain of D. seriata.
Inconsistency has also been observed, with some MBCAs being efficient in vitro but
less so in the field. It has been reported that their efficiency may sometimes depend on the
mode of application of the MBCAs in planta. Actually, Haidar et al. (2021) demonstrated
that the inhibitory activity of some bacterial strains against N. parvum was strongly affected
by the mode of application used, but had no effect on the efficacy of F. lateritium against
E. lata according to Munkvold and Marios (1993), using five potential biocontrol bacterial
strains against P. chlamydospora [82].
Formulation, the time of application, the phenological stage of the grapevine, its age,
and the cultivar may also affect the efficacy of MBCAs in vivo, as well as the origin of the
MBCAs [1].
The strains isolated from wood are more adapted to the physical and chemical con-
ditions of the grapevine wood [30]. For trials in vitro, the difference in efficiency may
also depend on the culture medium used in the experiment, as the medium’s chemical
composition may guide the metabolism of the bacterial and fungal MBCAs in one way or
another [121,122]. Drawing a parallel with these examples, Bardin et al. (2015) signaled
that this would likely occur for plant pathogens, especially when the biocontrol products
have a single mode of action. Therefore, these authors suggested that the hypothesis of
the “durability of biological control being higher than that of chemical control” may not
always be justified. Consequently, more research studies are required to anticipate the
integration of durability concerns in the screening procedure of new biocontrol agents [123].
To our knowledge, no resistance toward MBCAs by GTD fungi has been reported, which is
J. Fungi 2023, 9, 638 31 of 37

probably due to both their infrequent use and the complexity of their mechanisms of action;
however, this topic has not been studied enough.

8. General Discussion: Challenges and Prospects

The management of GTDs is difficult because (i) several pathogens are involved in
the same disease, (ii) the synergy between microorganisms degrades the wood [71], and
(iii) more than one trunk disease can sometimes occur in the same plant [2,42,124].
To control GTDs, the selection of MBCAs tolerant to pesticides and resistant to an-
timicrobials and toxins present in the environment may present a promising approach
to enhance their persistence and efficiency in the field. For instance, it was reported by
French et al. (2021) that the alternative use of MBCAs and conventional fungicides reduced
the levels of synthetic inputs and the risk of fungicide resistance. Using this strategy,
promising results were obtained with the strains T. atroviride, T. harzianum, and F. lateritium
that are benzimidazole-resistant [1,99,125], as well as Trichoderma strains (mainly T. afro-
harzianum and T. simmonsii) that are myclobutanil-resistant, when assessed against GTD
pathogens [31]. Another factor that has to be taken into account is the resistance of MBCAs
to the various antimicrobials and toxins in the environment, because they may affect their
persistence and biocontrol efficiency. For instance, Gkikas et al. (2021) evaluated the bio-
control potential of the strains P. alvei K165 rifampicin-resistant mutant and F. oxysporum
F2 hygromycin B-resistant mutant against P. chlamydospora. Accordingly, the selection of
MBCAs tolerant to pesticides and resistant to antimicrobials and toxins present in the
environment may present a promising approach to enhance their persistence and efficiency
in the field.
Another approach that is widely used in biocontrol to optimize impact is the de-
velopment of products containing multiple microbial strains with different modes of
action [126]. Currently, a product available on the market (i.e., Blindar) is based on
two Trichoderma strains, i.e., T. asperellum ICC012 and T. gamsii ICC080, which act by
antibiosis and competition for nutrients and space against GTD pathogens (Table 1). Re-
cently, Di Marco et al. (2022) demonstrated that the preventive application of this product
significantly reduced the expression of Esca symptoms under field conditions, and they
showed that its potential also persisted in the environment, as they re-isolated the two
Trichoderma strains after 7 months. A mixture of Trichoderma and Gliocladium was effective
in the field against Phaeoacremonium spp. and P. chlamydospora, but this was not the case
for a bacterial mixture of three strains of Azospirillum sp., Bacillus sp., and Pseudomonas sp.
against these pathogens under the same conditions [1]. In another study, no synergetic ef-
fect between P. oligandrum and P. agglomerans or B. pumilus was observed under greenhouse
conditions in controlling P. chlamydospora [53]. Martínez-Diz et al. (2021) demonstrated
that the combination of two or more beneficial MBCAs promoted the prevention of black
foot and Petri diseases in vineyards. Another relevant point, associated with the mixture
of biocontrol agents, is that the combination of beneficial microorganisms with different
modes of action reduces the probability of resistance development by the pathogens.
Improving the methods of strain selection is, therefore, crucial to optimize the effi-
ciency/persistence of the MBCAs. Temperature plays an important role in this regard. This
enables relevant pieces of information to choose the optimal time to apply the product in
the field. Another relevant issue to be checked is the ability of some endophytic species
efficient to control one pathogen, but potentially stimulate another disease. This observa-
tion was reported by Haidar et al. (2016) with the strain Bacillus sp. S43, which inhibited
Botrytis cinerea infection but increased the symptoms caused by N. parvum when applied on
grapevine cuttings. Hence, interest in conducting comparative screening bioassays is of the
utmost importance.

9. Conclusions
To control GTDs, it is now becoming increasingly clear that no single effective control
measure must be used, with disease management based on integrated strategies combining
J. Fungi 2023, 9, 638 32 of 37

various control methods such as physical, chemical, biological control, cultural practices,
and tolerant grapevine cultivars being on the rise. These integrated strategies also include
other techniques aimed at limiting the propagation of the pathogens and the infection
risk, mainly during the nursery process and upon the plantation/establishment of new
vineyards [8,127].
Another important point is that integrated management strategies to manage GTDs
respond to the societal demand for low-environmental-impact and ecofriendly strategies of
plant protection. As for integrated management strategies, the use of MBCAs to develop
durable and ecological products to manage this devastative disease is also on the rise.
However, the selection of useful strains remains a major challenge, especially with regard
to optimizing the efficiency and the persistence of the MBCAs in the field, whether they
consist of a single microbial strain or a mixture of strains. The taxa of MBCAs also play a
major role in the selection process, since some microbial species or genera are known to
produce toxins or to be potential plant pathogens (e.g., Fusarium and Erwinia). In addition,
the selection of strains able to grow on variable nutrient sources (mainly carbon and
nitrogen) may express a double advantage: a high potential for competition with pathogens
and a low cost of industrial production.
For the future, another key challenge will be to decipher the microbiome of grapevine,
since pathogens responsible for pathogenicity interact with the plant and its microbiome. It
is assumed that potential endophytic MBCAs isolated from grapevine are highly effective
against GTD fungi, because they are adapted to the wood tissue environment and they
share the same host as the pathogens [30,42]. For this reason, further studies aimed at
understanding the microbial interactions in the wood of diseased and healthy grapevine
would be a key point for selecting microbial strains able to fight GTD pathogens. Equally,
in the case of integrated pest management, the sensibility of potential biocontrol strains to
chemicals and their adaptability to nursery or field conditions, as well as the optimization
of product formulation, are relevant points to be studied.

Author Contributions: Writing—original draft preparation O.M. and P.R.; writing—review and
editing, O.M., R.H., A.Y., A.D.-Z., J.-Y.B., R.G., E.A. and P.R.; supervision and funding acquisition P.R.
and E.A. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Excellence Initiative of Université de Pau et des Pays
de l’Adour–I-Site E2S UPPA [Project Biovine, seed funding], a French “Investissements d0 Avenir”
program. The French Research Technology Association (ANRT), and the GreenCell Company. It
was also funded by the Industrial Chair “WinEsca” funded by the ANR (French National Research
Agency), as well as the JAs Hennessy & Co and GreenCell companies.
Conflicts of Interest: The authors declare no conflict of interest.

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