ArticlePDF AvailableLiterature Review

Abstract and Figures

Since 2013, Xylella fastidiosa Wells et al. has been reported to infect several hosts and to be present in different areas of Europe. The main damage has been inflicted on the olive orchards of southern Apulia (Italy), where a severe disease associated with X. fastidiosa subspecies pauca strain De Donno has led to the death of millions of trees. This dramatic and continuously evolving situation has led to European and national (Italian and Spanish) measures being implemented to reduce the spread of the pathogen and the associated olive quick decline syndrome (OQDS). Research has been also carried out to find solutions to better and directly fight the bacterium and its main insect vector, Philaenus spumarius L. In the course of this frantic effort, several treatments based on chemical or biological substances have been tested, in addition to plant breeding techniques and integrated pest management approaches. This review aims to summarize the attempts made so far and describe the prospects for better management of this serious threat, which poses alarming questions for the future of olive cultivation in the Mediterranean basin and beyond.
Content may be subject to copyright.
microorganisms
Review
Xylella fastidiosa in Olive: A Review of Control Attempts and
Current Management
Massimiliano Morelli 1, JoséManuel García-Madero 2,Ángeles Jos 3, Pasquale Saldarelli 1,
Crescenza Dongiovanni 4, Magdalena Kovacova 5, Maria Saponari 1, Alberto Baños Arjona 2,
Evelyn Hackl 6, Stephen Webb 5and Stéphane Compant 6,*


Citation: Morelli, M.; García-Madero,
J.M.; Jos, Á.; Saldarelli, P.;
Dongiovanni, C.; Kovacova, M.;
Saponari, M.; Baños Arjona, A.;
Hackl, E.; Webb, S.; et al. Xylella
fastidiosa in Olive: A Review of
Control Attempts and Current
Management. Microorganisms 2021,9,
1771. https://doi.org/10.3390/
microorganisms9081771
Academic Editors: Joana Costa, Joël
F. Pothier, Jens Boch, Emilio Stefani
and Ralf Koebnik
Received: 30 June 2021
Accepted: 14 August 2021
Published: 19 August 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1
Consiglio Nazionale delle Ricerche, Istituto per la Protezione Sostenibile delle Piante, Sede Secondaria di Bari,
70124 Bari, Italy; massimiliano.morelli@ipsp.cnr.it (M.M.); pasquale.saldarelli@ipsp.cnr.it (P.S.);
maria.saponari@ipsp.cnr.it (M.S.)
2DMC Research Center, 18620 Granada, Spain; jgmadero@dmcrc.com (J.M.G.-M.);
abarjona@dmcrc.com (A.B.A.)
3Área de Toxicología, Facultad de Farmacia, Universidad de Sevilla, 41012 Seville, Spain; angelesjos@us.es
4Centro di Ricerca, Sperimentazione e Formazione in Agricoltura Basile Caramia, 70010 Locorotondo, Italy;
enzadongiovanni@crsfa.it
5RTDS Group, 1080 Wien, Austria; maggiekovacova@gmail.com (M.K.); webb@rtds-group.com (S.W.)
6AIT Austrian Institute of Technology, Center for Health and Bioresources, 3430 Tulln, Austria;
evelyn.hackl@ait.ac.at
*Correspondence: stephane.compant@ait.ac.at
Abstract:
Since 2013, Xylella fastidiosa Wells et al. has been reported to infect several hosts and to be
present in different areas of Europe. The main damage has been inflicted on the olive orchards of
southern Apulia (Italy), where a severe disease associated with X. fastidiosa subspecies pauca strain
De Donno has led to the death of millions of trees. This dramatic and continuously evolving situation
has led to European and national (Italian and Spanish) measures being implemented to reduce the
spread of the pathogen and the associated olive quick decline syndrome (OQDS). Research has been
also carried out to find solutions to better and directly fight the bacterium and its main insect vector,
Philaenus spumarius L. In the course of this frantic effort, several treatments based on chemical or
biological substances have been tested, in addition to plant breeding techniques and integrated pest
management approaches. This review aims to summarize the attempts made so far and describe the
prospects for better management of this serious threat, which poses alarming questions for the future
of olive cultivation in the Mediterranean basin and beyond.
Keywords: bacterial disease; olive outbreak; sustainable control; IPM strategy; Philaenus spumarius
1. Introduction
Xylella fastidiosa Wells et al. (Xanthomonadaceae) [
1
] is a bacterial pathogen that has
been well documented for its worldwide spread and infection of a broad range of plant
species. The global distribution of this pathogen continues to increase due to anthropogenic
movements of goods and plant materials. Recent surveys have shown several incursions
(ingresses) of this pathogen in Europe and the Mediterranean basin [2].
Different subspecies of X. fastidiosa are known, which are described according to the
host range and genetic relationships [
3
]. Indeed, the bacterium is highly polyphagous,
infecting 638 plant species, and in several cases without causing symptoms [
4
]. Currently,
three subspecies of X. fastidiosa (fastidiosa,multiplex, and pauca) are generally accepted by
the scientific community to be the main grouping, although there is no type strain available
for subspecies pauca in the public databases [5]. Proposals also exist for the establishment
of the subspecies sandyii,morus, and tashke [
6
], and the situation is continuously evolving
due to the fast acquisition of new genomic information and strain distinction by multilocus
sequence typing [7].
Microorganisms 2021,9, 1771. https://doi.org/10.3390/microorganisms9081771 https://www.mdpi.com/journal/microorganisms
Microorganisms 2021,9, 1771 2 of 21
Among the new X. fastidiosa plant pathosystems, infections in olive plants during
recent years (Table 1) have had a very devastating outcome because of the severe symptoms
of olive quick decline syndrome (OQDS), which was first described in 2013 [
8
]. In suscepti-
ble cultivars, X. fastidiosa infestations have led to extensive desiccation, which results in
widespread tree die-off. Discovered in Salento, Apulia, Italy, X. fastidiosa subspecies pauca
strain “De Donno” ST53 [
9
,
10
] has quickly spread from its original infection zone, due to
the very efficient and highly prevalent vector Philaenus spumarius L., a xylem sap feeder
insect (Hemiptera: Aphrophoridae) [
11
]. In Italy, the spread of X. fastidiosa has been greatly
facilitated by favorable vector conditions, the planting of extensive monocultures of two
autochthonous susceptible olive cultivars (Cellina di Nardòand Ogliarola salentina), and
the current dense population of X. fastidiosa on infected olive trees [12].
Table 1.
Reported occurrence
1
of Xylella fastidiosa subspecies in olive (Olea europaea L.) trees in the EU.
Geographic Area Xylella fastidiosa Subspecies Sequence Type
Apulia, Italy pauca ST53
Provence-Alpes-Côte d’Azur, France pauca ST53
Provence-Alpes-Côte d’Azur, France multiplex Unknown
Ibiza, Balearic Islands, Spain pauca ST80
Mallorca, Balearic Islands, Spain multiplex ST81
Madrid Community, Spain multiplex ST6
Porto Metropolitan Area, Portugal multiplex ST7
1
Data are those reported in the latest release of the Xylella spp. host plant database [
4
] and updated to 31
December 2020.
Since its first detection, X. fastidiosa subspecies pauca strain “De Donno” has infected
about 4 million trees in the outbreak area [
13
]. This has caused, and is still causing,
enormous economic losses of olive trees and oil production outputs, in addition to dramatic
changes in the Mediterranean landscape, with olive trees being a strongly ingrained part
of the cultural heritage and an important element of the flourishing tourism industry.
Two years after its discovery in Apulia, X. fastidiosa was considered no longer eradicable
due to the large occurrence of infected plants in the outbreak area. This induced the
National and European Phytosanitary Authorities to move from an “eradication” to a
“containment” strategy [
14
]. Its recent detection in the mid-Apulian region close to the
area of monumental olive trees in 2020 [
15
] caused great public alarm because of the high
value of these millennial trees, which besides being protected by regional law, have been
proposed to have UNESCO heritage status.
Genetic studies of the X. fastidiosa strain “De Donno” suggest that this pathogenic
strain was introduced and not native to the Mediterranean region. Phylogenetic analysis
indicates that the “De Donno” strain is closely related to a strain of X. fastidiosa from Costa
Rica, which is capable of infecting oleander (Nerium oleander L.) and coffee (Coffea spp.)
plants. Based on this information, it is suspected that the introduction of X. fastidiosa
to the Mediterranean region resulted from the importation of ornamental plants [
16
,
17
].
Moreover, after performing an extensive study of the genomic diversity of several X.
fastidiosa subspecies, the introduction of the olive strain ST53 in Apulia was dated back to
2008 [18].
Xylella fastidiosa was first reported in Spain in 2016 in the Balearic Islands. Three
subspecies (multiplex,fastidiosa, and pauca) and 4 sequence types (multiplex ST81 and ST7,
fastidiosa ST1 and pauca ST 80) have infected more than 20 plant species, including grape
(Vitis spp.), almond (Prunus spp.), olive (Olea europaea L.), and fig (Ficus spp.), on all islands
except Formentera [
19
]. According to a recent study, 79.5% of the almond trees on the
islands (approx. 1,250,000 trees) have been infected by X. fastidiosa [
20
]. In the Valencian
Community, X. fastidiosa was first noticed in 2017 on almond trees (unidentified species).
In December 2020, more than 600 wild or cultivated olive trees were reported as being
positive for X. fastidiosa in the Balearic archipelago [
19
]. According to the Generalitat
Valenciana (the government of Valencian Autonomous Community), 18 plant species have
Microorganisms 2021,9, 1771 3 of 21
been infected so far, although 93.6% of the samples found infected were from almond
trees (Prunus dulcis (Mill.) D. A. Webb). In 2021, the total area declared infected across the
whole community reached 2292 ha., with more than 100,000 almond trees having been
destroyed [
21
]. In 2018, a single olive tree grown in the Madrid area was tested positive
for X. fastidiosa subsp. multiplex (ST6) [
22
]. In the same year, X. fastidiosa was detected
in three specimens of Polygala myrtifolia L. in a nursery of ornamental plants in Almeria
(Andalusia), although currently it is considered to be already eradicated at this site [20].
In late 2018, a new outbreak of X. fastidiosa in Italy was reported in Tuscany [
23
].
Multilocus sequence typing and genomic analyses of isolates infecting different ornamental
plants and almond trees revealed the occurrence of the newly discovered ST87, belonging
to subsp. multiplex and phylogenetically related to all the variants detected in the EU [
24
].
In France, X. fastidiosa subsp. multiplex has been reported in Corsica (2015), Provence (2019),
and Occitanie (2020). Lavender (Lavandula spp.), ornamental plants, myrtle shrub (Myrtus
communis L.), rosemary (Salvia rosmarinus Spenn.), and broom (Spartium junceum L.), as
well as two olive trees, were found to be infected by X. fastidiosa subsp. pauca, although this
was promptly eradicated. The outbreaks in the French regions seem to be due to several
introductions during the 1980s [25].
A more recent introduction occurred in 2019 in Portugal, where X. fastidiosa subsp.
multiplex ST7 was found in some asymptomatic plants of French lavender (Lavandula dentata
L.). Although more extensive surveys have led to the identification of about 50 other host
species within the country, so far only four olive trees have been found to be infected and
promptly uprooted [4].
As in other X. fastidiosa epidemics, such as Pierce’s disease (PD) of grapevine and citrus
variegated chlorosis, the current disease management approach relies on control activities
that integrate agronomical (soil tillage and weed elimination) and chemical interventions.
Measures aimed at reducing the vector populations involve the elimination of the sources
of inoculum, which in many places in Apulia are centenary olive trees protected by law [
26
];
however, such measures are currently not sufficient. The search for a cure for X. fastidiosa-
infected plants is an ongoing process to which not only academics, professionals, and
growers but also ordinary citizens continuously contribute (for example by searching for
spontaneous olive seedlings surviving from X. fastidiosa infection in the epidemic area).
Due to the severity of induced disease and the economic importance of the susceptible
crops, the discovery of an efficient solution would be self-promoting among people who
make a living from agriculture.
The present review lists most of the attempts made so far to combat X. fastidiosa in
olive plants, distinguishing between different types of control strategies used, including
containment or eradication measures taken by the EU and national governments, direct X.
fastidiosa and vector control, plant breeding, and integrated pest management approaches.
In addition, we report on X. fastidiosa infection in almond plants and the containment
measures implemented in Spain, because in this area, as in Italy, the bacterium is severely
affecting a major crop. The limited outbreaks identified in France and Portugal, which
have mainly occurred on ornamental plants, are not considered as they are outside the
scope of the present review. Prospects for better control are further discussed, as the threat
posed by X. fastidiosa to European agriculture, and particularly to olive, demands urgent
solutions. Additionally, the proposed solutions are bound to comply with the sustainability
principles expressed by the European Commission (EC) in the European Green Deal.
2. Containment and Eradication Measures
2.1. Xylella fastidiosa and EU Legislation
In response to the appearance of the X. fastidiosa outbreak in Italy, the EC took neces-
sary action as soon as possible to prevent the introduction and spread of X. fastidiosa by
enforcing the Commission Implementing Decision (EU) 2015/789 [
14
]. This legislative act
detailed contingency plans, the establishment of demarcated areas, containment measures,
eradication measures, and last but not the least, protection measures against X. fastidiosa.
Microorganisms 2021,9, 1771 4 of 21
The following year, in 2016, the EC decided to continue regulating X. fastidiosa within the
European Union as a quarantine pest under Regulation (EU) 2016/2031 [
27
], in continuity
with the previous Directive 2000/29 [28].
With the continuous spread of X. fastidiosa in Europe, on the 14th of August 2020,
the EC decided to repeal (EU) 2015/789 [
14
] by introducing the new Regulation (EU)
2020/1201 [
29
], which included new and adapted control measures based on the latest
findings from the European Food Safety Authority (EFSA). In summary, Regulation (EU)
2020/1201 [
29
] mandates that upon the detection of an outbreak due to X. fastidiosa in
any EU member state, the relevant plant health authorities within that member state shall,
without delay, establish a demarcated area, which includes an infected zone and a buffer
zone. According to article 4 (EU) 2020/1201 [
29
], the infected zone shall have a radius of at
least 50 m around the X. fastidiosa-infected plants. The buffer zone shall have a width of at
least 5 km in the case when an infected zone established is subject to containment measures,
as set out in articles 12 to 17; a width of at least 2.5 km in the case when an infected zone
is subject to eradication measures, as set out in articles 7 to 11; and a width of 1 km for
isolated X. fastidiosa outbreaks where eradication measures have been immediately taken
(Figure 1).
Figure 1. Measures of containment/eradication against Xylella fastidiosa.
The established width of 5 km for the infected zones within the demarcated areas
subject to containment measures has sparked discussions amongst scientists and farmers,
as this is an adapted measure from the previous Commission Implementing Decision (EU)
2015/789 [
14
]. It has caused concerns over its efficacy, as the 5 km buffer zone might not be
sufficient in containing the spread of X. fastidiosa, in comparison to the previously mandated
widths of 20 km (containment zone) and 10 km (buffer zone), as per (EU) 2015/789 [14].
Regarding eradication measures within articles 7 to 11 of (EU) 2020/1201 [
29
], all
infected or symptomatic plants have to be removed, including those that belong to the
same species as the infected plants (irrespective of their current plant health status), as well
as other plants susceptible to X. fastidiosa within the infected zone. All other host plants
within the 50 m infected zone must be sampled and tested for X. fastidiosa. Regarding the
2.5 km buffer zone, the member state’s relevant authority shall further test and sample the
Microorganisms 2021,9, 1771 5 of 21
host plants, including intensive surveillance. The surveys shall take into consideration the
EFSA Guidelines on Risk-Based and Statistically Sound Surveys [30].
Regulation (EU) 2020/1201 [
29
] also details specific containment measures under
articles 12 to 17 for regions where eradication of X. fastidiosa is not feasible. These include
Southern Apulia (Italy), Corsica (France), and the Balearic Islands (Spain). Within these
regions (and within the infected zones), all infected plants shall be removed followed
by intensive surveillance within an area measuring at least 5 km from the border of the
infected zone with the buffer zone. For the 5 km buffer zones, the eradication measures
apply (Figure 1).
Lastly, Regulation (EU) 2020/1201 [
29
] also includes measures related to the planting
of specified plants within the infected zone, the movement of plants within and out of the
demarcated areas, as well as control measures to prevent the introduction of X. fastidiosa
into the European Union.
With more findings being shared by EFSA and other EU research projects related to X.
fastidiosa, further adaptations of EU provisions regarding the spread of X. fastidiosa in the
EU territory can be expected in the near future.
2.2. Italy
Regarding EU regulations, and since the first detection of X. fastidiosa in Apulia in 2013,
several legislative decrees have been issued by the Italian Ministry of Agriculture, Food,
and Forestry to detail the emergency measures proposed to prevent, control, and eradicate
X. fastidiosa from the Italian territory, in compliance with EU Regulation 2015/789 [
14
] and
successive modifications. The application of measures is assigned to the Regional Phytosan-
itary Services operating in synergy with and referring to the Central Phytosanitary Service.
The basic principles translated in the Italian legislation are currently: (1) the distinction
of “host” (species susceptible to all X. fastidiosa subspecies worldwide) and “specified”
(species susceptible to the local X. fastidiosa genotype) plants; (2) control measures in Apulia,
which were soon changed from “eradication” to “containment”; (3) territory demarcation
in infected and buffer zones as detailed above. Based on these principles, the emergency
plan currently consists of monitoring plant infection status in the buffer zone and moni-
toring the insect vector (P. spumarius) life stage to plan agronomic and chemical control
measures. Moreover, the planting of susceptible species in the infected area is prohibited,
except for species or cultivars found to be resistant or tolerant, along with the prohibition
of movement of “host” and “specified” plants out of the demarcated area. The results of
monitoring, legislative documents, scientific advances, and other information are regularly
updated on the freely accessible Apulia regional website dedicated to the X. fastidiosa
emergency [31].
2.3. Spain
In Spain, the MAPA (Ministerio de Agricultura, Pesca y Alimentación) Contingency
Plan for X. fastidiosa was elaborated for the first time in 2015, which was revised for
the last time in October 2020 by the Ministry of Agriculture, Fisheries, and Food after
the publication of EU Regulation 2020/2021 [
32
]. The plan provides specific guidelines
regarding: (1) the organization and responsibilities of the interested groups involved in
the plan; (2) legal provisions for the pest, background, and symptoms; (3) relevant factors
regarding the prevention, detection, damage, and control of the pest; (4) containment and
eradication procedures, including official measures. Legal provisions to consider include
EU regulations, national laws, and autonomic regulations such as the ones established
in the Balearic Islands, Valencian Community, Community of Madrid, and Andalusia.
Competences in this plan are distributed among the MAPA, autonomous communities
(forest and plant health official bodies), their diagnostic laboratories, and also the national
reference laboratories.
Upon detection of an outbreak, the competent bodies of the autonomous communities
should establish an Emergency Management Team to deal with tactical and operational
Microorganisms 2021,9, 1771 6 of 21
aspects of the contingency plan or specific action plans. These plans are executed when
the pathogen/disease is detected as a result of a general inspection, specific surveys, when
competent authorities are informed of its presence by an operator or individual, or due to
import or movement of plants.
When an autonomous community suspects the presence of an outbreak of X. fastidiosa,
a series of precautionary measures included in the contingency plan must be further
adopted [
32
]. Once confirmed by the official laboratory or a national reference laboratory,
the detection of the outbreak should be reported to the MAPA and measures included in
the contingency plan to prevent the spread of the pathogen and to achieve its eradication
should be adopted. Moreover, the European Commission and the other member states
must also be informed. Demarcated areas are then established, delimiting an infected zone
and a buffer zone following the guidelines established in Regulation (EU) 2020/1201 [
29
]
and where eradication measures must be adopted. Additionally, the competent official
bodies (MAPA and the affected Autonomous Community) must establish a plan that
provides information on the disease and the contingency plan must be published on the
websites of these bodies. This information must be widely distributed to all stakeholders,
the general public, travelers, professionals, and international transport companies.
2.4. Considerations for Existing Measures in the Framework of the “Farm to Fork Strategy”
Following the recent EU and national provisions regarding X. fastidiosa, which rely
on the control of the vector and inoculum sources, the development of biopesticides to
target the bacterium and its vector is of paramount importance to help reduce the spread
of the pathogen in Europe. Most noteworthy is the reduced width of the infected zone in
areas where containment measures apply, which may facilitate the spread of the disease. In
light of such fears, the relevant authorities of the Apulia region have decided to maintain
the old demarcation areas as mandated by the initial Commission Implementing Decision
(EU) 2015/789 [
14
]. More studies and observations are needed to determine the efficacy
of the abovementioned decisions. As such, should the worst-case scenario become reality
and the newly adopted (EU) 2020/1201 [
29
] 5 km width of the infected zone prove not
to be efficient in preventing the spread of X. fastidiosa, the introduction of curative and
preventive solutions for X. fastidiosa-infected plants might be the only effective solution
regarding the containment and eradication of X. fastidiosa.
Once novel biopesticides are introduced and production is upscaled to the market,
the EU legislation on protective measures against X. fastidiosa might be more relaxed in the
near future.
Within the framework of the recently published Farm to Fork Strategy [
33
], the heart
of the European Green Deal, the EC is determined to reduce the use and risk of chemical
pesticides; thus, there is an urgent need to introduce low risk and non-chemical pesticides
as alternative means to deal with dangerous pathogens.
3. Current Attempts to Control X. fastidiosa in Olive—State of the Art
Since the first identification of X. fastidiosa strain “De Donno” in olive trees [
34
] and
the observation of the devastating damage caused in Apulian orchards by the associated
disease [
10
], researchers have produced a growing body of literature on the attempts to
control the pathogen, in addition to government measures, through the application of
different treatments [
35
]. An extensive literature search of the approaches that have been
taken within a fairly limited time returned a satisfactory number of experimental attempts,
including
in vitro
studies and the application of potential treatment solutions directly to
affected olive trees. Most experiments to control X. fastidiosa on olive trees have been
performed in Italy.
The different studies have relied on several control approaches, which in turn involve
mineral formulations, chemical compounds, natural products, and microbial antagonists
(Figure 2, top). It is worth noting that given the recent introduction of this highly virulent
strain of X. fastidiosa in Apulia, most of these studies are considered to be at the preliminary
Microorganisms 2021,9, 1771 7 of 21
stage, although in the absence of an effective strategy, some of these attempts appear to be
at least promising.
Figure 2. Current approaches to control Xylella fastidiosa epidemics.
3.1. Minerals and Compounds Control: Moving beyond Conventional Agrochemicals
A series of studies conducted
in vitro
[
36
38
] showed that alterations in mineral
homeostasis, mainly involving zinc, copper, and calcium ions, may have significant effects
on X. fastidiosa Temecula1, which is responsible for PD in grapevine, affecting relevant
biological features, such as the biofilm formation and growth rate, and possibly interfering
with the expression of its virulence traits in the host tissues. Based on these studies, recent
experiments aimed to investigate how the plant ionome, i.e., the relative content of mineral
elements found in a specific tissue [
39
], could interfere with the expression of symptoms
caused by the X. fastidiosa strain “De Donno” in olive trees. Interestingly, pursuing this
approach, D’Attoma et al. [
40
,
41
] provided evidence that higher contents of calcium and
manganese may contribute to resistance traits shown by the cultivar Leccino. Relying
on a similar strategy, Del Coco et al. [
42
] investigated the perturbation of the ionomic
profile of the leaves, following treatment of X. fastidiosa-infected trees with Dentamet
®
, a
zinc–copper–citric acid biocomplex. This study corroborated the evidence gathered from a
parallel approach that relied on a metabolomic analysis to reveal substantial changes in
the metabolic profiles of X. fastidiosa-susceptible olive cultivars, following crown treatment
with Dentamet
®
complex [
43
]. It should be noted that the earliest descriptions of the
application of Dentamet
®
via foliar spray had shown that this complex can reduce X.
fastidiosa-associated disease severity; however, the restricted time range of the application
and the limited number of observations did not allow for conclusive evidence of complete
eradication of the pathogen [
44
]. A further mid-term assessment revealed that the bacterial
concentration tended to decrease in trees regularly sprayed with the biocomplex over
3–4 years [45].
Aside from the administration of zinc and copper, other strategies to control X. fastid-
iosa in olive plants and employing mineral solutions have been attempted in Italy. When
Microorganisms 2021,9, 1771 8 of 21
sprayed with ammonium chloride, OQDS-affected trees showed clear symptom reduc-
tions; however, no significant differences in the bacterial populations were observed [
46
].
Recently, metal nanooxides have also been explored as carriers for the direct release of
phytodrugs targeting X. fastidiosa in olive plants. Transmission electron microscopy ob-
servations showed an alteration of the bacterial cell wall following the interactions with
calcium carbonate nanocarriers, which were absorbed by the olive roots and successfully
translocated to conductive tissues [47].
Among the most well-studied control strategies for X. fastidiosa is also N-acetylcysteine
(NAC). This mucolytic cysteine analogue, used mainly to treat human diseases [
48
], had
shown promising inhibitory effects on X. fastidiosa strain 9a5c and its associated disease in
sweet orange plants [
49
]. Building on this experience, some field trials were performed
in Apulia to verify the NAC effect on OQDS. In general, treatment with NAC seems
to decrease disease progression, especially using NAC endotherapy; however, qPCR
assays did not show any significant reduction in the bacterial population size [
50
]. When
investigating its effect on X. fastidiosa strain “De Donno” regarding
in vitro
behavior, Cattò
et al. [
51
] found, however, that sub-lethal concentrations of NAC had a significant effect
on X. fastidiosa biofilm formation, inducing a hyper-attaching phenotype, with potential
impacts on strain virulence and vector acquisition.
Other approaches to reduce X. fastidiosa involved fosetyl–aluminum nanocrystals
coated with chitosan [
52
] antimicrobial peptides (AMPs) [
53
], however, so far such mineral
solutions have not led to efficient X. fastidiosa disease control and further products are
still needed.
None of the mineral-based approaches have proven sufficient to control the bacterium
in planta; therefore, none of the approaches have been validated for use in the current man-
agement strategy. Consequently, no data exist regarding the development of X. fastidiosa
resistance to the applied minerals or on potential effects on the olive microbiota. As many
of these minerals or compounds have significant
in vitro
effects on the bacterium lifestyle
or survival, future research trends should consider optimizing their delivery to better target
X. fastidiosa in the xylem network.
3.2. Plant- and Microbial-Derived Compounds
To explore the use of natural products from plants or microorganisms, Bleve et al. [
54
]
evaluated the
in vitro
antimicrobial activities of different classes of plant-derived pheno-
lics compounds (4-methylcathecol, cathecol, veratric acid, caffeic acid, and oleuropein),
filtered fractions of olive mill wastewaters (OMW), Trichoderma spp. culture extracts, and
fungal toxins. All tested phenolic compounds showed some inhibitory activities against
X. fastidiosa strain “De Donno” isolated from olive plants, although limited to reversible
bacteriostatic effects. Moreover, ophiobolin A and gliotoxin showed bacteriostatic inhibi-
tion, whereas a crude culture extract from a strain of Trichoderma citrinoviridae exhibited
bactericidal properties. Interestingly, the addition of microfiltered OMW fractions in the
growth medium impacted “De Donno” growth.
Several phenolic compounds, such as coumarins, stilbenes, and flavonoids, have been
evaluated using
in vitro
assays for their potential use against PD-associated X. fastidiosa
strains. Overall, these phenolic compounds were effective in inhibiting X. fastidiosa growth,
as indicated by low minimum inhibitory concentrations. In addition, phenolic compounds
with different structural features exhibited different antagonistic capacities. Particularly,
catechol, caffeic acid, and resveratrol showed the highest inhibitory potential against the
pathogen [
55
]. In similar assays,
in vitro
activity was evaluated in different concentrations
of phenolic compounds, such as gallic acid, epicatechin, and resveratrol, on the growth of
X. fastidiosa. Although none of these compounds inhibited bacterial growth in a significant
way, some of them, such as epicatechin and gallic acid, reduced cell surface adhesion. In
addition, cell–cell aggregation decreased with resveratrol treatment [56].
Plant oils and extracts of multiple botanical species have also been tested. They are
among the constituents of NuovOlivo
®
, a natural bioactive detergent, which was also
Microorganisms 2021,9, 1771 9 of 21
tested for its effectiveness in controlling OQDS. Spray treatments based on a small number
of samples and a limited time span lowered the disease index and X. fastidiosa DNA levels,
in addition to inducing plant defense and protecting cell membrane integrity [
57
], although
more field assays should be carried out to sufficiently prove the potential of the product.
An attempt to reduce X. fastidiosa and its associated plant symptoms was made based
on its diffusible signal factors. The lifecycle of X. fastidiosa proved to be finely regulated by
a complex metabolic pathway regulated by a family of short-chain fatty acid molecules
known as diffusible signal factors (DSF) [
58
,
59
], whose potential application for biological
control of X. fastidiosa-associated diseases has been extensively investigated in grapevine
and citrus plants [
60
,
61
]. New experiments are now underway pursuing the chemical
characterization of DSF produced by the strain “De Donno”, in order to identify chemical
analogues to be used for modulating cell-to-cell signaling and to help reduce the impacts
of X. fastidiosa infections on olives [62].
3.3. Microbial Control of X. fastidiosa Infections
This plant hosts different microorganisms in its plant organs. Differences between
susceptible and resistant/tolerant varieties for a disease have been correlated to the plant
microbiome [
63
,
64
]. The unprecedented interest in microbial endophytes as biocontrol
agents against pathogens [
63
,
65
], together with some promising, although preliminary,
indications of their use in the control of strain Temecula1 [
66
68
], has boosted the quest for
similar solutions to be applied against X. fastidiosa strain “De Donno” and to mitigate the
effects of the associated disease.
Recently, two studies explored the potential role of microbial endophytes in contribut-
ing to the expression of resistance traits against OQDS, which was described with some
olive cultivars. Vergine et al. [
69
] observed an overt dysbiosis established by X. fastidiosa
infection in the susceptible cultivar Cellina di Nardò, which was not found in the resistant
cultivar Leccino, whose microbial communities showed a greater diversity. The tendency
of the endophytic microbiome to succumb to the occupation of the whole ecological niche
by X. fastidiosa as the infection progresses was confirmed in the analysis by Giampetruzzi
et al. [
70
], who found that this trend was more evident in the susceptible cultivar Kalamata
than in the resistant FS-17
®
. Albeit differences in the microbiomes of the susceptible ver-
sus resistant cultivars were observed, when evaluating the biocontrol potency of several
endophytic bacterial strains isolated from olive trees located in the X. fastidiosa-affected
area, none of them proved to be efficient in inhibiting “De Donno” growth [
71
]. Similar
efforts to identify potential biocontrol agents within the endophytic microbial communities
inhabiting X. fastidiosa-infected olive trees led, however, to the discovery of Methylobac-
terium mesophilicum and M. radiotolerans strains found to be able to produce extracellular
siderophores. Microorganisms producing these Fe
3+
-binding compounds can significantly
enhance their biocontrol efficiency [
72
]; therefore, investigations are being conducted to
evaluate in planta and
in vitro
growth competition assays and the effects of these naturally
occurring Methylobacterium strains on “De Donno” populations [73].
Among other microbial strains, the beneficial endophyte Paraburkholderia phytofirmans
PsJN, isolated from onion roots [
74
,
75
], is known to colonize several host plants
[75,76]
,
stimulating their growth and protecting them against biotic and abiotic stresses [
76
]. It has
been shown to be effective in reducing Pierce’s disease symptom severity and X. fastidiosa
Temecula1 populations in grapevines [
77
]. Preliminary trials aimed at testing its effective-
ness as a biocontrol agent in the “De Donno” olive pathosystem in Italy, although limited
to a single season, have not revealed significant differences in the reduction of OQDS
symptoms in therapeutic treatments, nor reduction of the new infections upon preventive
applications [
78
]. Other microbial strains are also currently being tested for control of X.
fastidiosa under field conditions; however, so far no validated microbial-based solution is
currently available for farmers, pinpointing that efforts need to also be directed toward
other approaches to reduce the pathogen load in olive trees.
Microorganisms 2021,9, 1771 10 of 21
4. Current Attempts to Control the Insect Vector(s) in Olives—State of the Art
Vector control is the principal method available for controlling many insect-borne dis-
eases [
79
]. The lack of therapeutic applications to cure plants infected by X. fastidiosa makes
vector control the main option available to limit the spread of the pathogen in contami-
nated areas (Figure 2, middle). Given the unique mechanism (persistent non-circulative
and without a latency period) underlying the insect transmission of X. fastidiosa and the
difficulties in interfering or disrupting the acquisition and transmission processes, vector
control aims to limit the transmission by reducing or eliminating the vector populations
visiting susceptible host plants.
4.1. Survey of the Insect Vectors
Surveys for potential insect vectors of X. fastidiosa in Europe and the Mediterranean
countries have unambiguously indicated spittlebugs (Hemiptera: Aphrophoridae), mainly
those belonging to the genus Philaenus, as the dominant xylem sap feeders in olive
groves [
80
]. Currently, the only confirmed vectors of X. fastidiosa in Europe are P. spumarius
(Hemiptera: Aphrophoridae), and under experimental conditions, Neophilaenus campestris
Fallén and Philaenus italosignus Drosopolous and Remane [
26
]. Before the emergence of
X. fastidiosa in Europe, these insect species were neglected and poorly investigated, as they
were never associated with significant direct damage to crops. Nowadays, because of their
primary role as the European vectors of X. fastidiosa, they have gained major attention from
the scientific community [
81
]. The outcomes of several studies are now available, disclosing
relevant information on their biology and ecology (e.g., feeding behavior, host preference,
bacterial transmission efficiency, population dynamics) and providing important hints to
implement effective control strategies [81,82].
Spittlebugs are univoltine, overwintering as eggs and with a development cycle con-
sisting of a pre-imaginal stage (five instars) before the emergence of the adults. Control
strategies can target both nymphal and adult populations, although applications targeting
the nymphs are more effective and sustainable. The nymphs have limited movement
ability, and under the current epidemiological scenarios in the European outbreaks, do not
contribute to the spread of the infections [
81
]. Adults can acquire and transmit the bac-
terium soon after feeding on infected plants (without a latency period) and throughout the
whole life span (i.e., from May to October, depending on the climatic conditions), thereby
challenging the effectiveness of chemical control to protect plants from theinfections during
the whole adult season.
Bodino et al. [
80
] described the stage-structured populations of nymphs and adults
in Apulian olive groves, allowing determination of the best time to apply control mea-
sures. According to their observations, the newly hatched nymphs (1st instar) always
disappeared just before the peak of the 4th instar nymphs, while the first adults were only
captured after this peak; therefore, any control measure applied at the 4th instar peak could
potentially target the whole nymph population before the onset of the adults, achieving
the maximum efficacy.
4.2. Weed Management
With regard to the specific interventions used to reduce the juvenile populations,
several trials have been conducted in Italy and Spain under laboratory, semi-field, and
field conditions. More specifically, Dongiovanni et al. [
83
] compared over three years the
effectiveness of different applications targeting weeds and ground vegetation as means
to reduce juveniles of P. spumarius and N. campestris. The following trials were compared:
(i) no tillage; (ii) soil tillage performed twice (in early winter and in spring when the
majority of the nymphs were at the IV instar); (iii) soil tillage performed only during winter;
(iv) sowing Poaceae crops (Lolium spp. L. and Hordeum vulgare L.); (v) shallow ploughing;
(vi) mulching; (vii) application of herbicides; (viii) pyroweeding. The population densities
recorded (nymphs/m
2
) in these trials clearly showed that regardless of the interventions,
all were able to significantly reduce the juveniles of both spittlebug species compared to
Microorganisms 2021,9, 1771 11 of 21
the non-disturbed ground vegetation (no tillage). More specifically, the use of a systemic
herbicide, pyroweeding, and double tillage yielded the highest efficacies for both spittlebug
species. Tillage performed in winter was effective for N. campestris but not for P. spumarius.
Sowing Poaceae proved to be consistently effective for P. spumarius, although yielded
inconsistent results for N. campestris (i.e., was efficient during the first year but not upon
the second year of sowing these species in the same experimental plot) [83].
4.3. Insecticide Use to Control the Vector
Besides interventions to remove the ground vegetation, different insecticides and
natural compounds directly targeting the nymphs have also been tested [
84
]. Field trials
conducted in Italy showed that among the products tested, neonicotinoids and pyrethroids
consistently caused significant reductions of nymph populations. The other products tested
(i.e., buprofenzin, sweet orange essential oil, kaolin, and zeolite) contributed to reductions
of the number of nymphs (compared to the non-treated ground vegetation) but at lower
effectiveness than the systemic insecticides. Additionally, the application of mineral oil (for
eggs) and pelargonic acid tested in a single trial led to the lowest mortality rates for both
spittlebug species [85].
Laboratory testing carried out in Spain [
84
] showed that pyrethroids (deltamethrin
and
λ
-cyhalothrin) provided good control of P. spumarius nymphs and were fast acting
(95% of mortality in 24 h). Sulfoxaflor (Closer
®
) exhibited similar efficacy at 48 and 72 h
but it was slow acting. In contrast, pymetrozine and spirotetramat caused low mortality
rates in nymphs. Higher efficacy was achieved when natural pyrethrins were combined
with 1% or 3% of piperonyl butoxide: the mortality rate increased from approximately
30% (pyrethrin alone) to 95% (pyrethrin+ piperonyl butoxide) regardless of the amount of
piperonyl butoxide added.
Overall, the testing of measures against the nymphal populations in Italy and Spain
confirmed pyrethroids as the most efficacious class of insecticides, in line with previous
data collected for the sharpshooter leafhopper Homalodisca vitripennis Germar (Hemiptera:
Cicadellidae) [
86
]. Indeed, these studies disclosed important information on several al-
ternative products, including synthetic and natural insecticides, which can be used in
combination or as alternatives to mechanical interventions for integrated management of
the spittlebug populations in areas where X. fastidiosa-infections occur or pose a serious
threat (i.e., buffer zones, areas close due to outbreaks, etc.). Regarding the control of adults,
different insecticides have also been tested on olives. The data available to date are mainly
from trials carried out in Italy [
87
], where testing has been performed under semi-field
conditions, i.e., by caging a pre-fixed number of adult spittlebugs on olive branches, with
mortality rates assessed at different times after spraying.
Similarly to the results recorded for nymphs, synthetic pyrethroids (deltamethrin
and lambda-cyhalothrin) and neonicotinoids (imidacloprid, acetamiprid, thiamethoxan)
showed the highest efficacy against adults, causing increased mortality (with rates rang-
ing from 76.7% to 100% at 3-DAT), with persistence up to 15–20 DAT (days after treat-
ments) for pyrethroids and 20–25 DAT for neonicotinoids. Organophosphorus insecticides
(chlorpyrifos-ethyl and chlorpyrifos-methyl) yielded lower mortality rates or inconsistent
results, except for the applications based on phosmet, which caused mortality rates ranging
from 69% to 82.5% and persistence up to 15 DAT [87].
Promising results were also obtained for cyantraniliprole (anthranilic diamide) [
88
].
The mortality and persistence rates were in some cases higher than those recorded for the
reference products (pyrethroids and neonicotinoids), whereas spinosad, abamectin, and
sweet orange essential oil showed prompt effects but poor or no persistence. Nevertheless,
no mortality was recorded after use of spirotetramat, flonicamid, pymetrozine, buprofenzin,
natural pyrethrin, or azadirachtin [83].
Izquierdo and Sabaté[
89
] also evaluated different synthetic insecticides (pyrethroids,
flupyradifuron, spirotetramat) against juveniles and adults of P. spumarius. Among the
pyrethroids, delthamethrin showed a high knockdown effect against both developmental
Microorganisms 2021,9, 1771 12 of 21
stages, although with limited persistence, while flupyradifuron showed a slower prompt
effect but higher persistence [
89
]. As with the data collected in Italy, the authors did not
register insect mortality upon applications based on spirotetramat.
In almost all experimental trials against the adults on olives, the tested formulations
were sprayed onto the canopy; however, information on the effectiveness of the insecticides
using other modes of application is very limited. Dongiovanni et al. [
88
] reported compar-
ative field trials where dimethoate and imidacloprid were supplied by spray applications
and trunk injections. The results showed (i) a slower effect of the formulations injected into
the trunk than the effect (mortality) recorded for the same formulation applied by spraying
and (ii) inconsistent data in terms of persistence; nevertheless, no significant increase in
the persistence of the active ingredients was recorded upon trunk injections, as would
be expected.
It is worth noting that neonicotinoids have been used in several experimental trials as
“terms of reference”, with the aim of finding alternative formulations showing similar levels
of efficacy, although their use (i.e., the three active ingredients clothianidin, imidacloprid,
and thiamethoxam) has been recently banned or almost prohibited in the EU (Regulations
2013/485 and 2018/783) [
90
,
91
]; therefore, more safe products should be developed to
reduce P. spumarius populations.
Appropriate timing of the applying insecticides is a crucial aspect for the effectiveness
of the control strategies against the vector. The results of surveys carried out in infected
Italian olive groves [
92
] confirmed that X. fastidiosa-positive spittlebugs are detected soon
after the emergence of the adults in early May, with the incidence increasing throughout the
season; therefore, for an effective and sustainable control strategy, efforts should be made in
the early phase of the adult season in an attempt to reduce as much as possible the number
of adults visiting the infected olive canopies. Moreover, Bodino et al. [
82
] recently showed
that the transmission efficiency may also increase in autumn, suggesting that spittlebugs
visiting olive canopies late in the season, even if seldom, could also contribute to the disease
spread. Nevertheless, inoculation events taking place late in the season can contribute
to spreading infections over higher distances [
80
]. Interestingly, the experimental results
gathered by Bodino et al. [
82
] showed that P. spumarius is not a very efficient vector if
compared to H. vitripennis, the most effective sharpshooter vector in North American
pathosystems, although population level and preference for olive plants can compensate
for low efficiency. As such, the authors concluded that it is even more crucial to greatly
reduce populations of this spittlebug in olive groves (preferably at the nymphal stage),
since the number of feeding insects is probably the most important factor determining
successful transmission and the quick spread of the pathogen.
4.4. Natural Enemies
Although lacking knowledge of potential natural enemies hampers the effective im-
plementation of biological control measures [
93
], surveys for natural enemies of spittlebugs
have been intensified during the past few years in Europe. Reis et al. [
94
] reported the oc-
currence of egg parasitoids in Portugal. More recently, the occurrence of the egg parasitoid
Ooctonus vulgatus Haliday was reported in Corsica [
95
]. The predation dynamics of the gen-
eralist predator Zelus renardii Kolenati on P. spumarius were reported by Liccardo et al. [
96
].
Furthermore, Molinatto et al. [
97
] reported the infestation of field-collected spittlebugs
by the parasitoid fly Verrallia aucta Fallén. Data for these natural enemy–vector interac-
tions are preliminary and additional studies are needed to fully understand the degree of
parasitoid–prey specificity to avoid non-target effects associated with any augmentative
biocontrol measure.
4.5. Insect Repellent
Although very limited, attempts to reduce the transmission events have also been
performed in olive groves in Italy [
88
]. Specifically, the effectiveness of kaolin was evaluated
in a new olive plantation in the X. fastidiosa-infected area to assess its impacts as a potential
Microorganisms 2021,9, 1771 13 of 21
insect repellent. Plants were protected (on a calendar basis) for three years during the
whole season when adults are present. Applications with imidacloprid were used as
reference insecticide control with respect to untreated control plants. The detection of
the first infections was delayed by 6 months in the kaolin-treated plants and by 2 years
in the imidacloprid-treated plants. Although a slower progression of the infections was
detected among the treatments, after 3 years from planting no differences were recorded
between treated and untreated plants. Even so, the delayed infections positively correlated
with symptom onset, i.e., first shoot dieback on the untreated plants appeared already
during the first year, while it was recorded after two years in the kaolin-treated plants and
after three years on the plants sprayed with imidacloprid [
88
]. Overall, this experiment
showed that the use of kaolin and imidacloprid did not prevent infections of healthy plants
exposed to infective spittlebugs, although amelioration of the impacts of the infections
was observed in the short period, probably as a consequence of the delayed infections and
lower numbers of transmission events on the treated plants. These results further highlight
the difficulties in finding an effective control for adult spittlebugs, reinforcing the need to
put in place measures for the control of the juvenile forms.
4.6. Pheromones and Volatiles as Control Measures
Within the framework of developing control measures to include in integrated pest
management (IPM) strategies, some studies have investigated innovative approaches, i.e.,
exploring the manipulation of the feeding and sexual behavior of P. spumarius. Although
pheromones are not common in spittlebugs and are unlikely to be used as monitoring and
control tools, recent studies on the structure of antennal sensilla of the spittlebug allowed
the identification of chemoreceptors [
98
] and provided evidence of the functionality of
antennal sensilla in P. spumarius adults [
99
]. In this latter study, the authors demonstrated
the capability of the male and female olfactory systems to selectively perceive a variety
of volatile compounds with a possible info-chemical role that may modulate P. spumarius
intra- and interspecific interactions; however, it should be pointed out that among the
50 compounds tested in this work, only four compounds elicited significantly different
responses between males and females, suggesting a general similarity between sexes in
terms of antennal sensitivity. In a similar study, the behavioral responses of P. spumarius to
a selection of essential oils and aromatic plants were investigated. The results showed good
correlations between the bioactivity of odor sources and the negative and positive host
preferences, demonstrating the capability of the peripheral olfactory systems of both sexes
to perceive volatile compounds [
100
]. Indeed, these bioassays clearly indicated that males
and females of P. spumarius respond differently to the same volatile blend. Although the
recorded behavioral effects need to be confirmed under field conditions, these results may
allow for semiochemical-based control strategies (i.e., push-and-pull or attract-and-kill
strategy) to be developed in the future and for the use of essential oils to be extended for
sustainable control of the X. fastidiosa vector.
Regarding the manipulation of sexual behavior, interesting investigations are ongoing
to characterize the emission of vibrational signals by males and females. The first detailed
description of the vibrational signals with specific roles within the mating behavior of
P. spumarius was reported by Avosani et al. [
101
]. The authors concluded that even if further
research is needed to identify an efficient signal and the most suitable strategy for field
application, these preliminary results open up the possibility to manipulate P. spumarius
behavior through artificial playbacks and for the future development of low environmental
impact control practices, for example by attracting males into a trap using a specific
vibrational signal.
5. Plant Breeding as a Sustainable Solution
Olive species have large (more than 900 cultivars) genetic and phenotypic variability in
the Mediterranean area [
102
]. As soon as the epidemic started spreading in Apulia, several
field observations indicated that while the more widely represented cultivars Ogliarola
Microorganisms 2021,9, 1771 14 of 21
salentina and Cellina di Nardòwere highly susceptible to X. fastidiosa, traits of resistance
were found in the cultivar Leccino. Indeed, in trees of this cultivar, a lower incidence of
infections and limited canopy desiccations were observed.
Studies of the plant response to the infection [
10
,
103
] showed that in xylem tissues of
field-grown Ogliarola salentina and Leccino olives: (i) the bacterium population size of
Leccino is 100 times lower (4
×
10
4
vs. 2
×
10
6
CFU/mL) than that of Ogliarola salentina;
(ii) Leccino resistance is reproducible in greenhouse with artificial X. fastidiosa infections,
while both susceptible cultivars show severe symptoms; (iii) genes associated with plant cell
wall remodelling are altered in both cultivars, while receptor-like kinases and proteins and
drought-associated genes are upregulated in Leccino and Ogliarola salentina, respectively.
Similar molecular interactions were observed in grapevine [
104
,
105
] and citrus [
106
,
107
]
infected by X. fastidiosa, and importantly receptor-like kinases are present in the PDR1 locus
(pathogen disease resistance locus 1), as identified in the X. fastidiosa-resistant Vitis arizonica
Engelm. [
108
,
109
]. The PDR1 trait was introgressed in V. vinifera L., and recently five bred
grapevine cultivars were released on the market, indicating breeding for resistance as a
promising strategy for the management of X. fastidiosa infections [110].
Leccino resistance in the field was confirmed in additional studies reporting a differen-
tial ionomer composition with respect to Ogliarola salentina [
41
]; an increase in quinic acid
(a lignin precursor) in the Leccino response to the infection [
12
]; a possible role of the xylem
anatomy in the resistance [
111
,
112
], as also reported in grapevine [
108
] and citrus [
113
];
and the role of biofilm and plant tylose response [
114
,
115
]. Further resistance traits were
found in the cultivar FS17
®
, characterized by low bacterial population size and limited
desiccations [
116
]. Several other olive cultivars are currently under evaluation regarding
susceptibility to infections in the field or artificial conditions within the framework of the
XF-ACTORS Project [
117
,
118
]. Long-term studies of resistance stability and crop yield are
lacking, while their envisaged epidemiological benefits were confirmed as being reduced
acquisition and transmission of the bacterium by P. spumarius when the inoculum source
was Leccino or FS17®[119].
A collaboration between the CNR-IPSP and an agronomist or olive oil producer is
ongoing to evaluate the adoption of the grafting of Leccino on the susceptible cultivars
Ogliarola salentina and Cellina di Nardòto save centennial trees within the framework
of Project ResiXO, funded by the Apulian regional authority. In the same project, olive
seedlings naturally grown in the epidemic area and surviving the infection have been
selected and are now under screening to confirm the observed resistance and to evaluate
their technological (i.e., olive and olive oil productions) characteristics, again pursuing a
strategy of breeding for resistance to cohabit with the bacterium (Figure 2, bottom).
Integrated Pest Management (IPM)
Since “there are still no risk reduction options that can remove the bacterium from the
plant in open field conditions “, the core of IPM strategies used to control X. fastidiosa, when
it is established in an area, relies on “early detection and rapid application of phytosanitary
measures, consisting among others of plant removal and vector control” [
26
]
(Figure 2
, bot-
tom). This declaration, therefore, evokes the two main pillars of “containment” measures
of X. fastidiosa in the Apulian outbreak, namely the legislative (phytosanitary) measures
and their application by means of an IPM strategy.
The crucial points of the IPM approach are a strategy of vector control, efficient and
prompt detection of the bacterium, the removal of infected plants in the buffer zone, and
the search for and adoption of resistant or tolerant species and cultivars. Fundamental
to the design and implementation of such an IPM approach has been the study of the X.
fastidiosa pathosystem. In-depth studies carried out within the framework of EU-funded
POnTE [
120
] and XF-ACTORS [
121
] projects, as well as several regional projects, have
allowed new and continuous knowledge to be generated on the biology and epidemiology
of the disease (i.e., host range of the local X. fastidiosa strain, ecology of the transmitting
vector(s), plant species and cultivar susceptibility to the bacterium) and diagnostic tools for
Microorganisms 2021,9, 1771 15 of 21
laboratory (qPCR, ELISA) and early detection using remote sensing technologies; however,
more effort is still needed to provide a comprehensive and sustainable solution. Tests of
different biocontrol agents, mineral, and chemicals to target X. fastidiosa or the insect vector
are needed, along with more plant breeding.
6. Conclusions and Future Prospects
In this review, we have described the history of X. fastidiosa infection in Europe, mainly
on olive trees, and the actions that so far have been taken to fight the outbreak. Several
solutions have been developed to reduce X. fastidiosa infection and to combat its insect
vectors, in addition to breeding new resistant plant lines and using several approaches
related to IPM. Although significant studies having been carried out, the X. fastidiosa disease
is still spreading in olive groves. Traditional olive farms may change to the cultivation of
other plants, with deep and detrimental impacts on the landscape, society, and European
cultural heritage. There is an urgent need to work on new strategies and solutions and to
examine and adapt EU and government regulations. We need further initiatives to develop
sustainable solutions that will be effective under different pedo-climatic conditions. The EU-
BBI-JU project BIOVEXO [
122
], in which academic and RTO partners, as well as industry
leaders, SMEs, and farmers work together, aims to develop sustainable solutions targeting
X. fastidiosa and its insect vector. Other projects dealing with basic and applied research on
X. fastidiosa are also needed. XF-ACTORS and POnTE have provided enormous amounts
of scientific knowledge; the Life Resilience [
123
] and CURE-XF [
124
] projects are currently
evaluating and developing solutions as well.
Joint efforts such as those exemplified by EU-wide initiatives and projects will certainly
lead to a better understanding of the aetiology of the X. fastidiosa disease and will provide a
solid basis for arriving at new, environmentally sustainable, and finally successful solutions.
This is in line with the ambitions set out in the European Green Deal, which aims to create
a healthier and more sustainable EU food system through a 50% reduction in the use of
chemicals and more hazardous pesticides by 2030. Presently, olive trees in Southern Europe
are still at risk, and the disease might continue to spread to further areas; hence, visionary
thinking and novel strategies are of utmost importance in all approaches to plant pest and
disease control. It could take years before effective solutions are found, and farmers may
have hard times ahead due to the disease burden affecting their crops and livelihoods.
Scientists, farmers, and industry leaders, as well as regional, national, and international
authorities, must join efforts and maximize their endeavors to save these olive trees and
ensure their continued survival.
Author Contributions:
All authors have written, read, and agreed to the published version of the
manuscript.
Funding:
This project has received funding from European Union’s Horizon 2020 research and
innovation program under grant agreement no. 727987 and from the Bio-Based Industries Joint
Undertaking (BBI-JU) under grant agreement no. 887281. The JU receives support from the European
Union’s Horizon 2020 research and innovation programme. Grant agreements no. 727987 and no.
887281 are “Xylella fastidiosa Active Containment Through a Multidisciplinary-Oriented Research
Strategy”—XF-ACTORS and “Biocontrol of Xylella and its Vector in Olive Trees for Integrated Pest
Management”—BIOVEXO, respectively.
Conflicts of Interest:
Magdalena Kovacova and Stephen Webb are employees at RTDS Group and
JoséManuel García-Madero and Alberto Baños Arjona work partly at DOMCA in Spain. The
organizations had no role in the analyses or interpretation of data or financial interest. The other
authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.
Microorganisms 2021,9, 1771 16 of 21
References
1.
Wells, J.M.; Raju, B.C.; Hung, H.-Y.; Weisburg, W.G.; Mandelco-Paul, L.; Brenner, D.J. Xylella fastidiosa gen. nov., sp. nov:
Gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp. Int. J. Syst. Evol. Microbiol.
1987
,37, 136–143.
[CrossRef]
2.
EPPO Global Database. Distribution of Xylella fastidiosa (XYLEFA). Available online: https://gd.eppo.int/taxon/XYLEFA/
distribution (accessed on 12 April 2021).
3.
Nunney, L.; Schuenzel, E.L.; Scally, M.; Bromley, R.E.; Stouthamer, R. Large-scale intersubspecific recombination in the plant-
pathogenic bacterium Xylella fastidiosa is associated with the host shift to mulberry. Appl. Environ. Microbiol.
2014
,80, 3025–3033.
[CrossRef]
4.
European Food Safety Authority (EFSA); Delbianco, A.; Gibin, D.; Pasinato, L.; Morelli, M. Update of the Xylella spp. host plant
database—Systematic literature search up to 31 December 2020. EFSA J. 2021,19, 6674. [CrossRef]
5.
Marcelletti, S.; Scortichini, M. Genome-wide comparison and taxonomic relatedness of multiple Xylella fastidiosa strains reveal the
occurrence of three subspecies and a new Xylella species. Arch. Microbiol. 2016,198, 803–812. [CrossRef]
6.
Burbank, L.P.; Ortega, B.C. Novel amplification targets for rapid detection and differentiation of Xylella fastidiosa subspecies
fastidiosa and multiplex in plant and insect tissues. J. Microbiol. Methods 2018,155, 8–18. [CrossRef]
7.
Maiden, M.C.J.; Bygraves, J.A.; Feil, E.; Morelli, G.; Russell, J.E.; Urwin, R.; Zhang, Q.; Zhou, J.; Zurth, K.; Caugant, D.A.
Multilocus sequence typing: A portable approach to the identification of clones within populations of pathogenic microorganisms.
Proc. Natl. Acad. Sci. USA 1998,95, 3140–3145. [CrossRef] [PubMed]
8.
Saponari, M.; Boscia, D.; Altamura, G.; Loconsole, G.; Zicca, S.; Datome, G.; Morelli, M.; Palmisano, F.; Saponari, A.; Tavano, D.
Isolation and pathogenicity of Xylella fastidiosa associated to the olive quick decline syndrome in southern Italy. Sci. Rep.
2017
,7,
17723. [CrossRef] [PubMed]
9.
Giampetruzzi, A.; Saponari, M.; Almeida, R.P.P.; Essakhi, S.; Boscia, D.; Loconsole, G.; Saldarelli, P. Complete genome sequence of
the olive-infecting strain Xylella fastidiosa subsp. pauca De Donno. Genome Announc. 2017,5, e00569-17. [CrossRef] [PubMed]
10.
D’Attoma, G.; Morelli, M.; De La Fuente, L.; Cobine, P.A.; Saponari, M.; de Souza, A.A.; De Stradis, A.; Saldarelli, P. Pheno-
typic characterization and transformation attempts reveal peculiar traits of Xylella fastidiosa subspecies pauca strain De Donno.
Microorganisms 2020,8, 1832. [CrossRef] [PubMed]
11.
Cornara, D.; Saponari, M.; Zeilinger, A.R.; De Stradis, A.; Boscia, D.; Loconsole, G.; Bosco, D.; Martelli, G.P.; Almeida, R.P.P.;
Porcelli, F. Spittlebugs as vectors of Xylella fastidiosa in olive orchards in Italy. J. Pest Sci. 2017,90, 521–530. [CrossRef]
12.
Luvisi, A.; Aprile, A.; Sabella, E.; Vergine, M.; Nicoli, F.; Nutricati, E.; Miceli, A.; Negro, C.; De Bellis, L. Xylella fastidiosa
subsp. pauca (CoDiRO strain) infection in four olive (Olea europaea L.) cultivars: Profile of phenolic compounds in leaves and
progression of leaf scorch symptoms. Phytopathol. Mediterr. 2017,56, 259–273.
13.
Schneider, K.; Van der Werf, W.; Cendoya, M.; Mourits, M.; Navas-Cortés, J.A.; Vicent, A.; Lansink, A.O. Impact of Xylella fastidiosa
subspecies pauca in European olives. Proc. Natl. Acad. Sci. USA 2020,117, 9250–9259. [CrossRef] [PubMed]
14.
European Commission. Commission Implementing Decision (EU) 2015/789 of 18 May 2015 as regards measures to prevent the
introduction into and the spread within the Union of Xylella fastidiosa (Wells et al.). Off. J. Eur. Union 2015,125, 36–53.
15. Sportelli, G. Xylella, morte annunciata per la Piana degli olivi monumentali? Olivo Olio 2020,10, 12–18.
16.
Loconsole, G.; Saponari, M.; Boscia, D.; Datome, G.; Morelli, M.; Martelli, G.P.; Almeida, R.P.P. Intercepted isolates of Xylella
fastidiosa in Europe reveal novel genetic diversity. Eur. J. Plant Pathol. 2016,146, 85–94. [CrossRef]
17.
Giampetruzzi, A.; Saponari, M.; Loconsole, G.; Boscia, D.; Savino, V.N.; Almeida, R.P.P.; Zicca, S.; Landa, B.B.; Chacón-Diaz, C.;
Saldarelli, P. Genome-wide analysis provides evidence on the genetic relatedness of the emergent Xylella fastidiosa genotype in
Italy to isolates from Central America. Phytopathology 2017,107, 816–827. [CrossRef]
18.
Vanhove, M.; Retchless, A.C.; Sicard, A.; Rieux, A.; Coletta-Filho, H.D.; De La Fuente, L.; Stenger, D.C.; Almeida, R.P.P. Genomic
diversity and recombination among Xylella fastidiosa subspecies. Appl. Environ. Microbiol. 2019,85, e02972-18. [CrossRef]
19.
Olmo, D.; Nieto, A.; Borràs, D.; Montesinos, M.; Adrover, F.; Pascual, A.; Gost, P.A.; Quetglas, B.; Urbano, A.; García, J.d.D.
Landscape epidemiology of Xylella fastidiosa in the Balearic Islands. Agronomy 2021,11, 473. [CrossRef]
20.
MAPA—Spanish Ministry of Agriculture Fisheries and Food. Xylella fastidiosa. Available online: https://www.mapa.gob.es/es/
agricultura/temas/sanidad-vegetal/organismos-nocivos/xylella-fastidiosa/ (accessed on 12 April 2021).
21.
Generalitat Valenciana. Situación Xylella fastidiosa Comunitat Valenciana, Marzo de 2021. Available online: https://agroambient.
gva.es/es/web/agricultura/xylella-fastidiosa#:~:text=Situaci%C3%B3n%20Xylella%20fastidiosa%20Comunitat%20Valenciana%
2C%20marzo%20de%202021 (accessed on 14 July 2021).
22.
Tihomirova-Hristova, L.; Pérez-Díaz, M.; Antón-Iruela, O.; Bielsa-Lozoya, S.; García-Gutiérrez, S.; Monterde, A.; Navarro, I.;
Montes Borrego, M.; Barbé, S.; Marco-Noales, E. Current situation after Xylella fastidiosa first outbreak in an olive grove in
mainland Spain. In Proceedings of the 2nd European Conference on Xylella fastidiosa: How Research Can Support Solutions,
Ajaccio, France, 29–30 October 2019.
23.
Marchi, G.; Rizzo, D.; Ranaldi, F.; Ghelardini, L.; Ricciolini, M.; Scarpelli, I.; Drosera, L.; Emanuele, G.; Capretti, P.; Surico, G. First
detection of Xylella fastidiosa subsp. multiplex DNA in Tuscany (Italy). Phytopathol. Mediterr. 2018,57, 363–364.
24.
Giampetruzzi, A.; Datome, G.; Zicca, S.; Abou Kubaa, R.; Rizzo, D.; Boscia, D.; Saldarelli, P.; Saponari, M. Draft Genome sequence
resources of three strains (TOS4, TOS5, and TOS14) of Xylella fastidiosa infecting different host plants in the newly discovered
outbreak in Tuscany, Italy. Phytopathology 2019,109, 1516–1518. [CrossRef]
Microorganisms 2021,9, 1771 17 of 21
25.
Soubeyrand, S.; de Jerphanion, P.; Martin, O.; Saussac, M.; Manceau, C.; Hendrikx, P.; Lannou, C. Inferring pathogen dynamics
from temporal count data: The emergence of Xylella fastidiosa in France is probably not recent. New Phytol.
2018
,219, 824–836.
[CrossRef] [PubMed]
26.
Bragard, C.; Dehnen-Schmutz, K.; Di Serio, F.; Gonthier, P.; Jacques, M.A.; Miret, J.A.J.; Justesen, A.F.; MacLeod, A.; Magnusson,
C.S.; Milonas, P. Update of the Scientific Opinion on the risks to plant health posed by Xylella fastidiosa in the EU territory. EFSA J.
2019,17. [CrossRef]
27.
European Commission. Regulation (EU) 2016/2031 of the European Parliament of the Council of 26 October 2016 on protective
measures against pests of plants, amending Regulations (EU) No 228/2013, (EU) No 652/2014 and (EU) No 1143/2014 of the
European Parliament and of the Council and repealing Council Directives 69/464/EEC, 74/647/EEC, 93/85/EEC, 98/57/EC,
2000/29/EC, 2006/91/EC and 2007/33/EC. Off. J. Eur. Union 2016,317, 4–104.
28.
European Commission. Council Directive 2000/29/EC of 8 May 2000 on protective measures against the introduction into
the Community of organisms harmful to plants or plant products and against their spread within the Community. Off. J. Eur.
Communities 2000,169, 1–112.
29.
European Commission. Commission implementing regulation (EU) 2020/1201 of 14 August 2020, as regards measures to prevent
the introduction into and the spread within the Union of Xylella fastidiosa (Wells et al.). Off. J. Eur. Union 2020,L 269, 2–39.
30.
Lázaro, E.; Parnell, S.; Civera, A.V.; Schans, J.; Schenk, M.; Abrahantes, J.C.; Zancanaro, G.; Vos, S.; European Food Safety
Authority. General Guidelines for Statistically Sound and Risk-Based Surveys of Plant Pests; Wiley Online Library: Hoboken, NJ, USA,
2020; 65p.
31. Emergenza Xylella. Available online: http://www.emergenzaxylella.it/ (accessed on 12 April 2021).
32.
MAPA—Spanish Ministry of Agriculture Fisheries and Food. Plan de Contingencia de Xylella fastidiosa (Well y Raju); Programa
Nacional para la Aplicación de la Normativa Fitosanitaria. 2021. Available online: https://www.mapa.gob.es/es/agricultura/
temas/sanidad-vegetal/xylellafastidiosa_contingencia_marzo2021_tcm30-525545.pdf (accessed on 16 August 2021).
33.
European Commission. Farm to Fork Strategy. Available online: https://ec.europa.eu/food/farm2fork_en (accessed on 12 April
2021).
34.
Saponari, M.; Boscia, D.; Nigro, F.; Martelli, G.P. Identification of DNA sequences related to Xylella fastidiosa in oleander, almond
and olive trees exhibiting leaf scorch symptoms in Apulia (Southern Italy). J. Plant Pathol. 2013,95, 668.
35.
Saponari, M.; Boscia, D. Recent advances on the control of Xylella fastidiosa and its vectors in olive groves: State of the art from
the ongoing Europe’s Horizon 2020 research programs. In Proceedings of the BIOCONTROL, 4th International Symposium on
Biological Control of Bacterial Plant Diseases, Viterbo, Italy, 9–11 July 2019. [CrossRef]
36.
Cruz, L.F.; Cobine, P.A.; De La Fuente, L. Calcium increases Xylella fastidiosa surface attachment, biofilm formation, and twitching
motility. Appl. Environ. Microbiol. 2012,78, 1321–1331. [CrossRef] [PubMed]
37.
Cobine, P.A.; Cruz, L.F.; Navarrete, F.; Duncan, D.; Tygart, M.; De La Fuente, L. Xylella fastidiosa differentially accumulates mineral
elements in biofilm and planktonic cells. PLoS ONE 2013,8, e54936. [CrossRef]
38.
Navarrete, F.; De La Fuente, L. Zinc detoxification is required for full virulence and modification of the host leaf ionomer by
Xylella fastidiosa.Mol. Plant-Microbe Interact. 2015,28, 497–507. [CrossRef]
39.
Salt, D.E.; Baxter, I.; Lahner, B. Ionomics and the study of the plant ionomer. Annu. Rev. Plant Biol.
2008
,59, 709–733. [CrossRef]
40.
D’Attoma, G.; Saldarelli, P.; De La Fuente, L.; Cobine, P. Ionomes of plants infected with vascular pathogens: Xylella fastidiosa as a
case study. In Proceedings of the Plant Health 2019, APS Annual Meeting, Cleveland, OH, USA, 3–7 August 2019. Paper 12629.
[CrossRef]
41.
D’Attoma, G.; Morelli, M.; Saldarelli, P.; Saponari, M.; Giampetruzzi, A.; Boscia, D.; Savino, V.N.; De La Fuente, L.; Cobine, P.A.
Ionomic differences between susceptible and resistant olive cultivars infected by Xylella fastidiosa in the outbreak area of salento,
italy. Pathogens 2019,8, 272. [CrossRef]
42.
Del Coco, L.D.; Migoni, D.; Girelli, C.R.; Angilè, F.; Scortichini, M.; Fanizzi, F.P. Soil and leaf ionomer heterogeneity in Xylella
fastidiosa subsp. pauca-infected, non-infected and treated olive groves in Apulia, Italy. Plants 2020,9, 760.
43.
Girelli, C.R.; Del Coco, L.; Scortichini, M.; Petriccione, M.; Zampella, L.; Mastrobuoni, F.; Cesari, G.; Bertaccini, A.; D’amico,
G.; Contaldo, N. Xylella fastidiosa and olive quick decline syndrome (CoDiRO) in Salento (southern Italy): A chemometric 1 H
NMR-based preliminary study on Ogliarola salentina and Cellina di Nardòcultivars. Chem. Biol. Technol. Agric.
2017
,4, 1–9.
[CrossRef]
44.
Scortichini, M.; Jianchi, C.; De Caroli, M.; Dalessandro, G.; Pucci, N.; Modesti, V.; L’Aurora, A.; Petriccione, M.; Zampella, L.;
Mastrobuoni, F. A zinc, copper and citric acid biocomplex shows promise for control of Xylella fastidiosa subsp. pauca in olive
trees in Apulia region (southern Italy). Phytopathol. Mediterr. 2018,57, 48–72.
45.
Tatulli, G.; Modesti, V.; Pucci, N.; Scala, V.; L’Aurora, A.; Lucchesi, S.; Salustri, M.; Scortichini, M.; Loreti, S. Further
in vitro
assessment and mid-term evaluation of control strategy of Xylella fastidiosa subsp. pauca in olive groves of Salento (Apulia, Italy).
Pathogens 2021,10, 85.
46.
Dongiovanni, C.; Fumarola, G.; Zicca, S.; Surano, A.; Di Carolo, M.; Datome, G.
In vitro
and
in vivo
effects of ammonium chloride
on Xylella fastidiosa subsp. pauca infecting olives. In Proceedings of the 3rd European Conference on Xylella fastidiosa and
XF-ACTORS Final Meeting, Online Event, 26–30 April 2021. [CrossRef]
Microorganisms 2021,9, 1771 18 of 21
47.
Baldassarre, F.; De Stradis, A.; Altamura, G.; Vergaro, V.; Citti, C.; Cannazza, G.; Capodilupo, A.L.; Dini, L.; Ciccarella, G.
Application of calcium carbonate nanocarriers for controlled release of phytodrugs against Xylella fastidiosa pathogen. Pure Appl.
Chem. 2020,92, 429–444. [CrossRef]
48.
Hafez, M.M.; Aboulwafa, M.M.; Yassien, M.A.; Hassouna, N.A. Activity of some mucolytics against bacterial adherence to
mammalian cells. Appl. Biochem. Biotechnol. 2009,158, 97–112. [CrossRef]
49.
Muranaka, L.S.; Giorgiano, T.E.; Takita, M.A.; Forim, M.R.; Silva, L.F.; Coletta-Filho, H.D.; Machado, M.A.; de Souza, A.A.
N-Acetylcysteine in agriculture, a novel use for an old molecule: Focus on controlling the plant–pathogen Xylella fastidiosa.PLoS
ONE 2013,8, e72937. [CrossRef] [PubMed]
50.
Alves de Souza, A.; Coletta-Filho, H.D.; Dongiovanni, C.; Saponari, M. N-acetyl-cysteine for controlling Xylella fastidiosa in
citrus and olive: Understanding the differences to improve management. In Proceedings of the 2nd European Conference on
Xylella fastidiosa: How Research Can Support Solutions, Ajaccio, France, 29–30 October 2019.
51.
Cattò, C.; De Vincenti, L.; Cappitelli, F.; Datome, G.; Saponari, M.; Villa, F.; Forlani, F. Non-Lethal Effects of N-Acetylcysteine on
Xylella fastidiosa strain De Donno biofilm formation and detachment. Microorganisms 2019,7, 656. [CrossRef]
52.
Baldassarre, F.; Tatulli, G.; Vergaro, V.; Mariano, S.; Scala, V.; Nobile, C.; Pucci, N.; Dini, L.; Loreti, S.; Ciccarella, G. Sonication-
assisted production of fosetyl-al nanocrystals: Investigation of Human toxicity and
in vitro
antibacterial efficacy against Xylella
fastidiosa.Nanomaterials 2020,10, 1174. [CrossRef]
53.
Baro, A.; Badosa, E.; Montesinos, L.; Feliu, L.; Planas, M.; Montesinos, E.; Bonaterra, A. Screening and identification of BP100
peptide conjugates active against Xylella fastidiosa using a viability-qPCR method. BMC Microbiol.
2020
,20, 229. [CrossRef]
[PubMed]
54.
Bleve, G.; Gallo, A.; Altomare, C.; Vurro, M.; Maiorano, G.; Cardinali, A.; D’Antuono, I.; Marchi, G.; Mita, G.
In vitro
activity of
antimicrobial compounds against Xylella fastidiosa, the causal agent of the olive quick decline syndrome in Apulia (Italy). FEMS
Microbiol. Lett. 2018,365, fnx281. [CrossRef] [PubMed]
55.
Maddox, C.E.; Laur, L.M.; Tian, L. Antibacterial activity of phenolic compounds against the phytopathogen Xylella fastidiosa.Curr.
Microbiol. 2010,60, 53–58. [CrossRef] [PubMed]
56.
Lee, S.A.; Wallis, C.M.; Rogers, E.E.; Burbank, L.P. Grapevine phenolic compounds influence cell surface adhesion of Xylella
fastidiosa and bind to lipopolysaccharide. PLoS ONE 2020,15, e0240101. [CrossRef]
57.
Bruno, G.L.; Cariddi, C.; Botrugno, L. Exploring a sustainable solution to control Xylella fastidiosa subsp. pauca on olive in the
Salento Peninsula, Southern Italy. Crop Prot. 2020,139, 105288.
58.
Beaulieu, E.D.; Ionescu, M.; Chatterjee, S.; Yokota, K.; Trauner, D.; Lindow, S. Characterization of a diffusible signaling factor
from Xylella fastidiosa.MBio 2013,4, e00539-12. [CrossRef]
59.
Chatterjee, S.; Newman, K.L.; Lindow, S.E. Cell-to-cell signaling in Xylella fastidiosa suppresses movement and xylem vessel
colonization in grape. Mol. Plant-Microbe Interact. 2008,21, 1309–1315. [CrossRef]
60.
Lindow, S.; Newman, K.; Chatterjee, S.; Baccari, C.; Iavarone, A.T.; Ionescu, M. Production of Xylella fastidiosa diffusible signal
factor in transgenic grape causes pathogen confusion and reduction in severity of Pierce’s disease. Mol. Plant-Microbe Interact.
2014,27, 244–254. [CrossRef]
61.
Caserta, R.; Souza-Neto, R.R.; Takita, M.A.; Lindow, S.E.; De Souza, A.A. Ectopic expression of Xylella fastidiosa rpfF conferring
production of diffusible signal factor in transgenic tobacco and citrus alters pathogen behavior and reduces disease severity. Mol.
Plant-Microbe Interact. 2017,30, 866–875. [CrossRef]
62.
Vona, D.; Datome, G.; Cicco, S.; Morelli, M.; Saldarelli, P.; Saponari, M.; Farinola, G. Monitoring of biofilm production in Xylella
fastidiosa strain De Donno via biochemical signalling modulation. In Proceedings of the 2nd European Conference on Xylella
fastidiosa: How Research Can Support Solutions, Ajaccio, France, 29–30 October 2019.
63.
Mitter, B.; Brader, G.; Pfaffenbichler, N.; Sessitsch, A. Next generation microbiome applications for crop production—Limitations
and the need of knowledge-based solutions. Curr. Opin. Microbiol. 2019,49, 59–65. [CrossRef]
64.
Compant, S.; Cambon, M.C.; Vacher, C.; Mitter, B.; Samad, A.; Sessitsch, A. The plant endosphere world–bacterial life within
plants. Environ. Microbiol. 2021,23, 1812–1829. [CrossRef]
65.
Morelli, M.; Bahar, O.; Papadopoulou, K.K.; Hopkins, D.L.; Obradovi´c, A. Editorial: Role of endophytes in plant health and
defense against pathogens. Front. Plant Sci. 2020,11, 1312. [CrossRef] [PubMed]
66.
Rolshausen, P.; Roper, C.; Maloney, K. Greenhouse Evaluation of Grapevine Microbial Endophytes and Fungal Natural
Products for control of Pierce’s Disease; Final Report for CDFA Agreement Number: 16-0512-SA. 2017. Available online:
https://www.semanticscholar.org/paper/Final-Report-for-CDFA-Agreement-Number%3A-16-0512-SA-Rolshausen/f41a7
3b56fd4a19ca03397345a909eeefb5c6097 (accessed on 14 July 2021).
67.
Rolshausen, P.; Roper, C.; Kirkpatrick, B.; Cooksey, D.; Borneman, J.; Maloney, K. Control of Pierce’s Disease with fungal
endophytes of grapevines antagonistic to Xylella fastidiosa. In Proceedings of the Pierce’s Disease Research Symposium, San
Diego, CA, USA, 15–17 December 2010; pp. 224–228.
68.
Kirkpatrick, B.; Jones, D.-D.; Civerolo, E.; Purcell, A.H. Characterize and assess the biocontrol potential of bacterial endophytes of
grapevines in California. In Proceedings of the Pierce’s Disease Research Symposium, Coronado, CA, USA, 8–11 December 2003.
69.
Vergine, M.; Meyer, J.B.; Cardinale, M.; Sabella, E.; Hartmann, M.; Cherubini, P.; De Bellis, L.; Luvisi, A. The Xylella fastidiosa-
resistant olive cultivar “Leccino” has stable endophytic microbiota during the Olive Quick Decline Syndrome (OQDS). Pathogens
2020,9, 35. [CrossRef] [PubMed]
Microorganisms 2021,9, 1771 19 of 21
70.
Giampetruzzi, A.; Baptista, P.; Morelli, M.; Cameirao, C.; Neto, T.L.; Costa, D.; Datome, G.; Abou Kubaa, R.; Altamura, G.;
Saponari, M.; et al. Differences in the endophytic microbiome of olive cultivars infected by Xylella fastidiosa across Seasons.
Pathogens 2020,9, 723. [CrossRef] [PubMed]
71.
Zicca, S.; De Bellis, P.; Masiello, M.; Saponari, M.; Saldarelli, P.; Boscia, D.; Sisto, A. Antagonistic activity of olive endophytic
bacteria and of Bacillus spp. strains against Xylella fastidiosa.Microbiol. Res. 2020,236, 126467. [CrossRef] [PubMed]
72.
Susi, P.; Aktuganov, G.; Himanen, J.; Korpela, T. Biological control of wood decay against fungal infection. J. Environ. Manag.
2011,92, 1681–1689. [CrossRef]
73.
Antelmi, I.; Sion, V.; Lucchese, P.; Nigro, F. Methylobacterium spp., endophytes of olive trees, as potential biocontrol agents of
Xylella fastidiosa subsp. pauca. In Proceedings of the 2nd European Conference on Xylella fastidiosa: How Research Can Support
Solutions, Ajaccio, France, 29–30 October 2019.
74.
Sessitsch, A.; Coenye, T.; Sturz, A.V.; Vandamme, P.; Barka, E.A.; Salles, J.F.; Van Elsas, J.D.; Faure, D.; Reiter, B.; Glick, B.R.
Burkholderia phytofirmans sp. nov., a novel plant-associated bacterium with plant-beneficial properties. Int. J. Syst. Evol. Microbiol.
2005,55, 1187–1192. [CrossRef] [PubMed]
75.
Compant, S.; Kaplan, H.; Sessitsch, A.; Nowak, J.; Ait Barka, E.; Clément, C. Endophytic colonization of Vitis vinifera L. by
Burkholderia phytofirmans strain PsJN: From the rhizosphere to inflorescence tissues. FEMS Microbiol. Ecol.
2008
,63, 84–93.
[CrossRef] [PubMed]
76.
Mitter, B.; Petric, A.; Shin, M.W.; Chain, P.S.G.; Hauberg-Lotte, L.; Reinhold-Hurek, B.; Nowak, J.; Sessitsch, A. Comparative
genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies
with host plants. Front. Plant Sci. 2013,4, 120. [CrossRef]
77.
Baccari, C.; Antonova, E.; Lindow, S. Biological control of Pierce’s disease of grape by an endophytic bacterium. Phytopathology
2019,109, 248–256. [CrossRef]
78.
Morelli, M.; Dongiovanni, C.; Datome, G.; Giampetruzzi, A.; Loconsole, G.; Montilon, V.; Altamura, G.; Angione, D.; Saponari,
M.; Saldarelli, P. Assessment of Paraburkholderia phytorfirmans PsJN biocontrol potential against Xylella fastidiosa ‘De Donno’
strain in olive. In Proceedings of the 2nd European Conference on Xylella fastidiosa: How Research Can Support Solutions,
Ajaccio, France, 29–30 October 2019.
79.
Wilson, A.L.; Courtenay, O.; Kelly-Hope, L.A.; Scott, T.W.; Takken, W.; Torr, S.J.; Lindsay, S.W. The importance of vector control
for the control and elimination of vector-borne diseases. PLoS Negl. Trop. Dis. 2020,14, e0007831. [CrossRef]
80.
Bodino, N.; Cavalieri, V.; Dongiovanni, C.; Saladini, M.A.; Simonetto, A.; Volani, S.; Plazio, E.; Altamura, G.; Tauro, D.; Gilioli, G.;
et al. Spittlebugs of Mediterranean olive groves: Host-plant exploitation throughout the year. Insects
2020
,11, 130. [CrossRef]
[PubMed]
81.
Cornara, D.; Bosco, D.; Fereres, A. Philaenus spumarius: When an old acquaintance becomes a new threat to European agriculture.
J. Pest Sci. 2018,91, 957–972. [CrossRef]
82.
Bodino, N.; Cavalieri, V.; Pegoraro, M.; Altamura, G.; Canuto, F.; Zicca, S.; Fumarola, G.; Almeida, R.; Saponari, M.; Dongiovanni,
C.; et al. Temporal dynamics of Xylella fastidiosa subsp. pauca vector transmission to olive plants. Entomol. Gen.
2021
,117,
9250–9259.
83.
Dongiovanni, C.; Di Carolo, M.; Fumarola, G.; Tauro, D.; Altamura, G.; Cavalieri, V. Evaluation of insecticides for the control of
juveniles of Philaenus spumarius L., 2015–2017. Arthropod Manag. Tests 2018,43, tsy073. [CrossRef]
84.
Dáder, B.; Viñuela, E.; Moreno, A.; Plaza, M.; Garzo, E.; Del Estal, P.; Fereres, A. Sulfoxaflor and natural Pyrethrin with Piperonyl
Butoxide are effective alternatives to Neonicotinoids against juveniles of Philaenus spumarius, the european vector of Xylella
fastidiosa.Insects 2019,10, 225. [CrossRef] [PubMed]
85. POnTE Project Deliverable 9.1: Practical Solution for Management and Containment of Xf. Available online: https://ec.europa.
eu/research/participants/documents/downloadPublic?documentIds=080166e5cdd715c6&appId=PPGMS (accessed on 12 April
2021).
86.
Prabhaker, N.; Castle, S.J.; Toscano, N.C. Susceptibility of immature stages of Homalodisca coagulata (Hemiptera: Cicadellidae) to
selected insecticides. J. Econ. Entomol. 2006,99, 1805–1812. [CrossRef]
87.
Dongiovanni, C.; Altamura, G.; Di Carolo, M.; Fumarola, G.; Saponari, M.; Cavalieri, V. Evaluation of efficacy of different
insecticides against Philaenus spumarius L., vector of Xylella fastidiosa in olive orchards in Southern Italy, 2015–17. Arthropod Manag.
Tests 2018,43, tsy034. [CrossRef]
88.
Dongiovanni, C.; Fumarola, G.; Di Carolo, M.; Tedone, B.; Ancona, S.; Palmisano, F.; Silletti, M.; Cavalieri, V. Ulteriori acquisizioni
per il controllo di Philaenus spumarius vettore di Xylella fastidiosa. In Proceedings of the Giornate Fitopatologiche, Online Event, 27
October–12 November 2020; pp. 257–266.
89.
Izquierdo, J.; Sabaté, J.; Eficacia de deltametrín y flupiradifurona en el control de Philaenus spumarius. Phytoma. 2018, pp. 68–73.
Available online: https://www.researchgate.net/publication/330540913_Eficacia_de_deltametrin_y_flupiradifurona_en_el_
control_de_Philaenus_spumarius (accessed on 14 July 2021).
90.
European Commission. Commission Implementing Regulation (EU) No 485/2013 of 24 May 2013 amending Implementing
Regulation (EU) No 540/2011, as regards the conditions of approval of the active substances clothianidin, thiamethoxam and
imidacloprid, and prohibiting the use and sale of seeds treated with plant protection products containing those active substances.
Off. J. Eur. Union L 2013,139, 12–26.
Microorganisms 2021,9, 1771 20 of 21
91.
European Commission. Commission Implementing Regulation (EU) 2018/783 of 29 May 2018 amending Implementing Regula-
tion (EU) No 540/2011 as regards the conditions of approval of the active substance imidacloprid. OJ L 2018,61, 31–34.
92.
Cavalieri, V.; Altamura, G.; Fumarola, G.; di Carolo, M.; Saponari, M.; Cornara, D.; Bosco, D.; Dongiovanni, C. Transmission of
Xylella fastidiosa subspecies pauca sequence type 53 by different insect species. Insects 2019,10, 324. [CrossRef]
93.
Di Serio, F.; Bodino, N.; Cavalieri, V.; Demichelis, S.; Di Carolo, M.; Dongiovanni, C.; Fumarola, G.; Gilioli, G.; Guerrieri, E.;
Picciotti, U. Collection of data and information on biology and control of vectors of Xylella fastidiosa.EFSA Support. Publ.
2019
,16.
[CrossRef]
94.
Reis, C.; Villa, M.; Rodrigues, I.; Cameirão, C.; Baptista, P.; Pereira, J.A. Potential natural biocontrol agents of Aphrophoridae
eggs. In Proceedings of the 2nd Joint Annual Meeting “European Research on Emerging Plant Diseases”, Valencia, Spain, 23–26
October 2018; p. 79.
95.
Mesmin, X.; Chartois, M.; Genson, G.; Rossi, J.-P.; Cruaud, A.; Rasplus, J.-Y. Ooctonus vulgatus (Hymenoptera, Mymaridae), a
potential biocontrol agent to reduce populations of Philaenus spumarius (Hemiptera, Aphrophoridae) the main vector of Xylella
fastidiosa in Europe. PeerJ 2020,8, e8591. [CrossRef] [PubMed]
96.
Liccardo, A.; Fierro, A.; Garganese, F.; Picciotti, U.; Porcelli, F. A biological control model to manage the vector and the infection
of Xylella fastidiosa on olive trees. PLoS ONE 2020,15, e0232363. [CrossRef] [PubMed]
97.
Molinatto, G.; Demichelis, S.; Bodino, N.; Giorgini, M.; Mori, N.; Bosco, D. Biology and prevalence in Northern Italy of Verrallia
aucta (Diptera, Pipunculidae), a parasitoid of Philaenus spumarius (Hemiptera, Aphrophoridae), the main vector of Xylella fastidiosa
in Europe. Insects 2020,11, 607. [CrossRef] [PubMed]
98.
Ranieri, E.; Ruschioni, S.; Riolo, P.; Isidoro, N.; Romani, R. Fine structure of antennal sensilla of the spittlebug Philaenus spumarius
L. (Insecta: Hemiptera: Aphrophoridae). I. Chemoreceptors and thermo-/hygroreceptors. Arthropod Struct. Dev.
2016
,45, 432–439.
[CrossRef] [PubMed]
99.
Germinara, G.S.; Ganassi, S.; Pistillo, M.O.; Di Domenico, C.; De Cristofaro, A.; Di Palma, A.M. Antennal olfactory responses of
adult meadow spittlebug, Philaenus spumarius, to volatile organic compounds (VOCs). PLoS ONE
2017
,12, e0190454. [CrossRef]
100.
Ganassi, S.; Cascone, P.; Di Domenico, C.; Pistillo, M.; Formisano, G.; Giorgini, M.; Grazioso, P.; Germinara, G.S.; De Cristofaro,
A.; Guerrieri, E. Electrophysiological and behavioural response of Philaenus spumarius to essential oils and aromatic plants. Sci.
Rep. 2020,10, 3114. [CrossRef]
101.
Avosani, S.; Franceschi, P.; Ciolli, M.; Verrastro, V.; Mazzoni, V. Vibrational playbacks and microscopy to study the signalling
behaviour and female physiology of Philaenus spumarius.J. Appl. Entomol. 2021,145, 518–529. [CrossRef]
102.
Muzzalupo, I.; Stefanizzi, F.; Perri, E. Evaluation of olives cultivated in southern Italy by simple sequence repeat markers.
HortScience 2009,44, 582–588. [CrossRef]
103.
Giampetruzzi, A.; Morelli, M.; Saponari, M.; Loconsole, G.; Chiumenti, M.; Boscia, D.; Savino, V.N.; Martelli, G.P.; Saldarelli, P.
Transcriptome profiling of two olive cultivars in response to infection by the CoDiRO strain of Xylella fastidiosa subsp. pauca.
BMC Genom. 2016,17, 475. [CrossRef]
104.
Zaini, P.A.; Nascimento, R.; Gouran, H.; Cantu, D.; Chakraborty, S.; Phu, M.; Goulart, L.R.; Dandekar, A.M. Molecular profiling of
Pierce’s disease outlines the response circuitry of Vitis vinifera to Xylella fastidiosa infection. Front. Plant Sci.
2018
,9, 771. [CrossRef]
105. Choi, H.-K.; Iandolino, A.; da Silva, F.G.; Cook, D.R. Water deficit modulates the response of Vitis vinifera to the Pierce’s disease
pathogen Xylella fastidiosa.Mol. Plant-Microbe Interact. 2013,26, 643–657. [CrossRef] [PubMed]
106.
Rodrigues, C.M.; de Souza, A.A.; Takita, M.A.; Kishi, L.T.; Machado, M.A. RNA-Seq analysis of Citrus reticulata in the early stages
of Xylella fastidiosa infection reveals auxin-related genes as a defense response. BMC Genom.
2013
,14, 676. [CrossRef] [PubMed]
107.
De Souza, J.B.; Almeida-Souza, H.O.; Zaini, P.A.; Alves, M.N.; de Souza, A.G.; Pierry, P.M.; da Silva, A.M.; Goulart, L.R.; Dandekar,
A.M.; Nascimento, R. Xylella fastidiosa subsp. pauca Strains Fb7 and 9a5c from Citrus display differential behavior, secretome, and
plant virulence. Int. J. Mol. Sci. 2020,21, 6769.
108.
Deyett, E.; Rolshausen, P.E. Temporal dynamics of the sap microbiome of grapevine under high Pierce’s disease pressure. Front.
Plant Sci. 2019,10, 1246. [CrossRef] [PubMed]
109.
Riaz, S.; Huerta-Acosta, K.; Tenscher, A.C.; Walker, M.A. Genetic characterization of Vitis germplasm collected from the
southwestern US and Mexico to expedite Pierce’s disease-resistance breeding. Theor. Appl. Genet.
2018
,131, 1589–1602. [CrossRef]
110.
Quinton, A. UC Davis Releases 5 New Wine Grape Varieties. Available online: https://www.ucdavis.edu/food/news/uc-davis-
releases-five-new-wine-grape-varieties (accessed on 12 April 2021).
111.
Sabella, E.; Luvisi, A.; Aprile, A.; Negro, C.; Vergine, M.; Nicolì, F.; Miceli, A.; De Bellis, L. Xylella fastidiosa induces differential
expression of lignification related-genes and lignin accumulation in tolerant olive trees cv. Leccino. J. Plant Physiol.
2018
,220,
60–68. [CrossRef]
112.
Montilon, V.; Boscia, D.; Savino, V.; Saldarelli, P.; De Stradis, A. Evaluation of vascular occlusions in xylem vessels of olive
cultivars infected with Xylella fastidiosa. In Proceedings of the 2nd European Conference on Xylella fastidiosa: How Research
Can Support Solutions, Ajaccio, France, 29–30 October 2019.
113.
Niza, B.; Coletta-Filho, H.; Merfa, M.; Takita, M.; De Souza, A. Differential colonization patterns of Xylella fastidiosa infecting
citrus genotypes. Plant Pathol. 2015,64, 1259–1269. [CrossRef]
114.
De Benedictis, M.; De Caroli, M.; Baccelli, I.; Marchi, G.; Bleve, G.; Gallo, A.; Ranaldi, F.; Falco, V.; Pasquali, V.; Piro, G. Vessel
occlusion in three cultivars of Olea europaea naturally exposed to Xylella fastidiosa in open field. J. Phytopathol.
2017
,165, 589–594.
[CrossRef]
Microorganisms 2021,9, 1771 21 of 21
115.
Cardinale, M.; Luvisi, A.; Meyer, J.B.; Sabella, E.; De Bellis, L.; Cruz, A.C.; Ampatzidis, Y.; Cherubini, P. Specific fluorescence in
situ hybridization (FISH) test to highlight colonization of xylem vessels by Xylella fastidiosa in naturally infected olive trees (Olea
europaea L.). Front. Plant Sci. 2018,9, 431. [CrossRef] [PubMed]
116.
Boscia, D.; Altamura, G.; Ciniero, A.; Di Carolo, M.; Dongiovanni, C.; Fumarola, G.; Giampetruzzi, A.; Greco, P.; La Notte, P.;
Loconsole, G. Resistenza a Xylella fastidiosa in diverse cultivar di olivo. L’inf. Agrar. 2017,11, 59–63.
117.
Saponari, M.; Altamura, G.; Abou Kubaa, R.; Montilon, V.; Saldarelli, P.; Specchia, F.; Palmisano, F.; Silletti, M.R.; Pollastro, P.;
Zicca, S.; et al. Further acquisition on the response of a large number of olive cultivars to infections caused by Xylella fastidiosa
subsp. pauca, ST53. In Proceedings of the 2nd European Conference on Xylella fastidiosa: How Research Can Support Solutions,
Ajaccio, France, 29–30 October 2019.
118.
XF-ACTORS Project: Screening of Olive Cultivars for Searching Sources of Resistance to Xylella fastidiosa. Available online:
https://www.xfactorsproject.eu/screening-cultivars-resistance-xf/ (accessed on 12 April 2021).
119.
Cavalieri, V.; Dongiovanni, C.; Altamura, G.; Tauro, D.; Ciniero, A.; Morelli, M.; Bosco, D.; Saponari, M. Evaluation of olive cultivar
effect on the efficiency of the acquisition and transmission of Xylella fastidiosa by Philaenus spumarius (Hemiptera: Aphrophoridae).
In Proceedings of the 3rd Hemipteran-Plant Interactions Symposium (HPIS 2017), Madrid, Spain, 4–8 June 2017; p. 39.
120.
POnTE Project (Pest Organisms Threatening Europe). Available online: https://www.ponteproject.eu/ (accessed on 12 April
2021).
121.
XF-ACTORS Project (Xylella Fastidiosa Active Containment through a Multidisciplinary-Oriented Research Strategy). Available
online: https://www.xfactorsproject.eu/ (accessed on 12 April 2021).
122.
BIOVEXO Project (Biocontrol of Xylella and Its Vector in Olive Trees for Integrated Pest Management). Available online:
https://biovexo.eu/ (accessed on 12 April 2021).
123. Life Resilience Project. Available online: http://www.liferesilience.eu/home-eng/ (accessed on 12 April 2021).
124.
Cure XF Project (Capacity Building and Raising Awareness in Europe and in Third Countries to Cope with Xylella fastidiosa).
Available online: http://www.cure-xf.eu/ (accessed on 12 April 2021).
... At the beginning of November 2016, 15 outbreaks were recorded in the French Riviera area of Provence and two new host plants (Spartium junceum and Lavandula angustifolia) were identified . Moreover, X. fastidiosa was confirmed in 2019 in Provence (southwest), and then in Occitania (south) in 2020 on lavender hybrid plants, as well as on Afghan lavender and Jerusalem sage (European Food Safety Authority et al., 2022b;Morelli et al., 2021). ...
... Owing to its endophytic nature, full control of X. fastidiosa remains challenging, as the ability of available bactericides and mineral-based compounds to access xylem vessels where the pathogen establishes is limited (EFSA Panel on Plant Health et al., 2019a; Kyrkou et al., 2018;Montesinos et al., 2022;Morelli et al., 2021;Tatulli et al., 2022). Thus, new chemical compounds are required to achieve effective disease suppression. ...
Article
Full-text available
Xylella fastidiosa is xylem-limited bacterium capable of infecting a wide range of host plants, resulting in Pierce's disease in grapevine, citrus variegated chloro-sis, olive quick decline syndrome, peach phony disease, plum leaf scald, alfalfa dwarf, margin necrosis and leaf scorch affecting oleander, coffee, almond, pecan, mul-berry, red maple, oak, and other types of cultivated and ornamental plants and forest trees. In the European Union, X. fastidiosa is listed as a quarantine organism. Since its first outbreak in the Apulia region of southern Italy in 2013 where it caused devastating disease on Olea europaea (called olive leaf scorch and quick decline), X. fastidiosa continued to spread and successfully established in some European countries (Corsica and PACA in France, Balearic Islands, Madrid and Comu-nitat Valenciana in Spain, and Porto in Portugal). The most recent data for Europe indicates that X. fastidiosa is present on 174 hosts, 25 of which were newly identified in 2021 (with further five hosts discovered in other parts of the world in the same year). From the six reported subspecies of X. fastidiosa worldwide, four have been recorded in European countries (fastidiosa, mul-tiplex, pauca, and sandyi). Currently confirmed X. fas-tidiosa vector species are Philaenus spumarius, Neophi-laenus campestris, and Philaenus italosignus, whereby only P. spumarius (which has been identified as the key vector in Apulia, Italy) is also present in Americas. X. fastidiosa control is currently based on pathogen-free propagation plant material, eradication, territory demarcation , and vector control, as well as use of resistant plant cultivars and bactericidal treatments.
... Chemical curative control against the bacterium is still under study as zinc-copper-citric acid biocomplex has been shown to de-55 crease bacterium population in olive trees [42][43]. Otherwise prevention by use of resistant varieties, hygienic and cultural 56 measures (i.e., cover plant management), biological (i.e., parasitoids or spiders) and chemical (neonicotinoids and pyrethroids) 57 vector control are the pathways to achieve it [35,37]. Combining multiple of these control strategies is considered as the best 58 management strategy [27]. ...
... In the plot A, after the tillage, there was no overall effect of the factor treatment, but there was a crossover interaction. In fact, one of the methods to decrease short-range spreading of X. fastidiosa is the control of the vectors such as nymphs or newly 149 emerged adults (e.g., removal of ground vegetation) [29,37,51,57]. 150 In this study we assessed the effect of the management of the cover vegetation (i.e., mowing and tillage) in olive and vineyard 151 organic orchards to decrease the density of nymphs of X. fastidiosa vectors. Our results indicated that tillage and mowing could 152 be an efficient method for mechanical control since the nymphal density decreased between 10 to 50 % in the treatment plots 153 compared to the control ones. ...
Preprint
Full-text available
Xylella fastidiosa Wells (1987) (Proteobacteria:Xanthomonadaceae) is a xylem pathogen bacterium transmitted by xylem feeder insects that causes several important plant diseases such as Pierce’s disease in grapes or leaf scorch in almond and olives trees. The bacterium was detected in the Balearic Islands in October 2016, including three subspecies: fastidiosa , multiplex and pauca . The major potential vectors described in the Balearics are Philaenus spumarius L. and Neophilaenus campestris Fallen (1805). In order to interfere the life cycle of vectors, we tested the effect of mechanical control of the plant cover on the most vulnerable phases, such as nymphs and/or newly emerged adults. For this, we selected four organic orchards in Mallorca, three olive and one vineyard plots. Owners of each selected plot conducted mechanical control according to their common procedures and their own machinery, which in general included cut and tillage of the plant cover during March-April. Nymph abundance per surface (30 sampling points/treatment/orchard x 0,25 m2) was measured in each plot in a weekly basis before and after mechanical control. Our results indicated that either tillage and mowing decreased nymphal density of X. fastidiosa vectors in both types of crops. These results contribute to the integrated pest management of vectors by conducting feasible farm-based management of the regular plant cover. Abstract Figure
... The suppression of vector populations is pivotal for the control of vector-borne diseases, and the system P. spumarius-X. fastidiosa is no exception [24,151]. Mechanical control of nymphs and insecticides-based control of pre-infective adults of P. spumarius are only partially effective and the use of chemicals is associated to unwanted side effects. ...
Article
Full-text available
Philaenus spumarius is a cosmopolitan species that has become a major threat to European agriculture being recognized as the main vector of the introduced plant pathogen Xylella fastidiosa , the agent of the “olive quick decline syndrome”, a disease which is devastating olive orchards in southern Italy. Wolbachia are bacterial symbionts of many insects, frequently as reproductive parasites, sometime by establishing mutualistic relationships, able to spread within host populations. Philaenus spumarius harbors Wolbachia , but the role played by this symbiont is unknown and data on the infection prevalence within host populations are limited. Here, the Wolbachia infection rate was analyzed in relation to the geographic distribution and the genetic diversity of the Italian populations of P . spumarius . Analysis of the COI gene sequences revealed a geographically structured distribution of the three main mitochondrial lineages of P . spumarius . Wolbachia was detected in half of the populations sampled in northern Italy where most individuals belonged to the western-Mediterranean lineage. All populations sampled in southern and central Italy, where the individuals of the eastern-Mediterranean lineage were largely prevalent, were uninfected. Individuals of the north-eastern lineage were found only in populations from the Alps in the northernmost part of Italy, at high altitudes. In this area, Wolbachia infection reached the highest prevalence, with no difference between north-eastern and western-Mediterranean lineage. Analysis of molecular diversity of COI sequences suggested no significant effect of Wolbachia on population genetics of P . spumarius . Using the MLST approach, six new Wolbachia sequence types were identified. Using FISH, Wolbachia were observed within the host’s reproductive tissues and salivary glands. Results obtained led us to discuss the role of Wolbachia in P . spumarius , the factors influencing the geographic distribution of the infection, and the exploitation of Wolbachia for the control of the vector insect to reduce the spread of X . fastidiosa .
... However, the future of this cultivation is in peril due to climate change [2] as well as other reasons such as olive crop diseases (e.g. Xylella fastidiosa) [3]. Thus, there is a need to acquire and process a high volume and quantity of information related to the olive crop for taking immediate actions that will allow sustainable and perpetual production of high yield and quality. ...
Article
Various Remote Sensing (RS) technologies and platforms have been widely used in olive cultivation studies over the last 16 years. These technologies and platforms have been applied throughout the olive cultivation cycle, providing significant insights into olive growth and productivity. The goal of this review was to determine the importance of RS technologies and platforms in specific agronomic focus areas in order to determine the equipment and platform requirements in olive cultivation studies. For this reason, frequency and correspondence studies were carried out. Unmanned aerial vehicles, multispectral sensors, and vigour assessment found to be important in olive cultivation studies. Additionally, each agronomic focus area in an olive cultivation study presents different needs in equipment, proximity of sensing, and coverage area, indicating that this must be taken into account during field experiments with RS. Further technological improvements will permit the use of other RS technologies and platforms during future studies. Finally, future studies are expected to focus more on RS data processing as well as on the use of unmanned aerial and ground vehicles in swarms for data collection and performance of actions.
Article
Full-text available
The Apulia (southern Italy) ornamental sector has been facing regulatory obligations and trade limitations due to a Xylella fastidiosa (Xf) outbreak since 2013. Alternative options to encounter these constraints include the implementation of novel and sustainable ornamental production (NSM) practices. In this context, the purpose of this study is to assess simultaneously the environmental implications and economic viability of these options versus the conventional production options (CMs) among eight ornamental species (Abelia grandiflora, Bougainvillea cv Don Mario, Lantana camara cv Bandana rosa, Jasminum officinalis, Photinia fraseri cv Red Robin, Loropetalum chinense cv Black Pearl, Trachelospermum jasminoides, Viburnum lucidum). Life cycle assessment (LCA) and cost–benefit analysis (CBA) were used for this purpose. LCA revealed that NSM induced relatively less environmental impacts at the nursery level towards agricultural land occupation, climate change, fossil depletion, and water depletion. CBA showed that NSM increases moderately nursery business profitability in an economic sustainable way. An overall annual average gross margin of about EUR 192/1000 plants can be generated using NSM over the CM model. In general, this research provides a useful decision-support, helping nursery growers under the pressure of the threat of quarantine pests such as Xf to adopt NSM practices, which could be useful to produce ornamental and landscape plants with high sanitary quality.
Article
Full-text available
Given the importance of olive growing, especially in Mediterranean countries, it is crucial that there is a constant process of modernization aimed at both environmental sustainability and the maintenance of high standards of production. The use of remote sensing (RS) allows intervention in a specific and differentiated way in olive groves, depending on their variability, in managing different agronomic aspects. The potentialities of the application of RS in olive growing are topics of great agronomic interest to olive growers. Using the tools provided by RS and the modernization of the olive sector can bring great future prospects by reducing costs, optimizing agronomic management, and improving production quantity and quality. This article is part of a review that aims to cover the past, from the 2000s onwards, and the most recent applications of aerial RS in olive growing in order to be able to include research and all topics related to the use of RS on olive trees. As far as the use of RS platforms such as satellites, aircraft, and unmanned aerial vehicles (UAVs) as olive growing is concerned, a literature review showed the presence of several works devoted to this topic. This article covers purely agronomic matters of interest to olive farms (and related research that includes the application of RS), such as yielding and managing diseases and pests, and detection and counting of olive trees. In addition to these topics, there are other relevant aspects concerning the characterization of the canopy structure of olive trees which is particularly interesting for mechanized pruning management and phenotyping.
Article
Full-text available
Xylella fastidiosa subsp. pauca (Xfp) is the plant pathogenic bacterium causing the epidemic of olive quick decline syndrome decimating olive trees in the Apulia region (southern Italy). The lack of any effective therapeutic application for the control of this pathogen and its categorization as a regulated quarantine pathogen in many countries worldwide, impose mandatory eradication and containment measures. Based on current EU legislation, containment measures apply in those areas where the bacterium is widely established, such as in the Apulia region, and thus containment strategies to mitigate and cope with the infections are needed. We set up a field trial to assess if pruning interventions could limit and/or recover Xfp-infected trees by reducing the systemic spread of the bacterium and the severity of the desiccation phenomena typically compromising the crown of the highly susceptible cultivars, e.g., cv. Cellina di Nardò. Trees subjected either to major or light pruning interventions, including the removal of all the symptomatic branches, did not demonstrate a reduced bacterial colonization or development of symptoms. After two years of targeted pruning interventions, no significant amelioration of the sanitary status of the infected olive trees was recorded, suggesting that the sole application of these interventions is not effective to counteract the impact of the bacterium in the susceptible olive trees.
Article
Xylella fastidiosa is a pathogen that causes fatal plant diseases and damage to horticultural crops. Establishing the basic parameters is necessary to assess the risk of disease outbreaks as there are concerns about the spread of X. fastidiosa. This is done by analyzing the climatic characteristics and distribution patterns of X. fastidiosa and related insect vectors. In this study, we aimed to derive the common climatic characteristics of X. fastidiosa and three major insect vectors by using a statistical density function for four climatic factors. In addition, the distance between the occurrence areas was calculated spatiotemporally and classified into natural and anthropogenic spread. The optimal climatic conditions identified for X. fastidiosa and the insect vectors were similar, suggesting a high potential for X. fastidiosa spread when both occur in a neighborhood area. X. fastidiosa spread mostly depends on anthropogenic pathways, but natural spread by insect vectors could increase. This study provides necessary insights for the risk assessment of X. fastidiosa spread based on climate similarity and spread patterns.
Article
Abstract Systemic bacteria such as Xylella fastidiosa can be vertically transmitted by using disease propagative material during the nursery tree production. We tested the hypothesis that X. fastidiosa is not transmitted by asymptomatic olive tree sprouts. In addition, we investigated the distribution of bacteria in olive plants (different parts of canopy and root system) with initial leaf scorch symptoms and in conditions of symptoms spread into the whole canopy. In both studies, the presence of bacteria was tested by quantitative real‐time PCR (qRT‐PCR). For the first hypothesis, sprouts from an asymptomatic olive tree were rooted and checked for X. fastidiosa infection 32 months later. A total of 57.74% (41 of 71) rooted seedlings were positive for X. fastidiosa (Cycle threshold ‐ CT from 24.25 to 31.82) but only one plant had scorched leaves. Regarding the bacteria distribution into the disease plants, results based on qRT‐PCR shown that X. fastidiosa was systemically distributed through the olive plants canopies (30 to 77% of sampled tissues, with CT values from 19.56 to 31.59). Samples taken from the OQDS symptomatic leaves to three meters above as also in the root system were positive to presence of X. fastidiosa. The results obtained demonstrate that vegetative material from olive plants used for rooting must be carefully selected for the absence of X. fastidiosa. Also, based on the qRT‐PCR positive results, pruning could have a limited effectiveness to completely eliminate the bacterium from diseased plants, even from the ones showing low severity symptoms of olive quick decline syndrome.
Article
The bacterial pathogen Xylella fastidiosa continues to threaten agricultural production of many different crops around the world, with significant economic burden from crop loss, disease management, and surveillance costs. In addition to direct economic consequences, plant diseases caused by X. fastidiosa have had significant societal impacts in the most affected regions. Although X. fastidiosa infects long-term perennial crops and landscape plants, there has never been a truly effective treatment for plants once they become infected. This review discusses the threat of X. fastidiosa to agriculture, landscapes, and global commerce, in addition to the most recent progress in treatment for X. fastidiosa in infected plants. Current disease mitigation options include nutritional, chemical, biocontrol, and plant resistance-based strategies, with the potential to develop combined management approaches. Overall, several strategies are promising for disease suppression but there is still opportunity for innovation, especially in treatment approaches that can be administered systemically by soil or foliar spray routes. Prevention of severe disease outcomes and crop loss, and the ability to suppress pathogen populations and reduce transmission without heavy reliance on insecticides would have significant economic and environmental benefits.
Conference Paper
Full-text available
Ammonium salts are ionic compounds, soluble in water and strongly dissociated. Although with a different mode of action, timing and doses, they are widely used as artificial fertilizers, food additives, antimicrobial agents in textile industry, surfactants, disinfectants, antistatic agents and cleansing agents. This study aims to investigate the impact of ammonium chloride on the growth performance of Xylella fastidiosa strain De Donno and explore its potential use in the field to mitigate the impact of infections in olives. Three concentrations of ammonium chloride (0.25-0.5-1%) were tested in liquid medium to evaluate the influence on the bacterial viability after three days and six days of growth. Crystal violet assay was used to determine the effect of ammonium chloride on the adhesion of the bacterium and its ability to form biofilm. Gradual growth inhibition was observed with increasing concentration of ammonium chloride in the growth medium. The growth inhibition effect is evident on planktonic cells, but is significantly more pronounced on biofilm-forming cells due to the peculiar adhesiveness of the X. fastidiosa strain De Donno. Field experiments started only recently (2019-2020) and included applications of ammonium chloride 4 times/year from March to October, in olive groves with different incidence of infections. A general increase of the vegetation mass and an attenuation of the wilting and desiccation phenomena (particularly in the bottom portion of the canopies) was observed in all treated trees, although not supported by statistically significant differences. Quantitative PCR tests on the experimental plants did not reveal any difference in the population size of treated and untreated plants. In conclusion, ammonium chloride showed in vitro effects on bacterial growth/biofilm formation, and attenuation of symptoms severity was detected, although prolonged experiments and observations are needed. 17 3 | In vitro and in vivo effects of ammonium chloride on Xylella fastidiosa, subsp. pauca infecting olives
Article
Full-text available
Following a request from the European Commission, EFSA was asked to create and regularly update a database of host plant species of Xylella spp. Complying with an extension of the previous mandate, which now covers the period 2021-2026, the current version of Xylella spp. host plant database updates the previous release dated April 2020. Informative data have been extracted from 86 recent publications retrieved through an extensive literature search. This report is related to the fourth version of the database published in Zenodo in the EFSA Knowledge Junction community, covering articles selected from: a systematic literature review conducted up to 31 December 2020, Europhyt outbreak notifications up to 18 March 2021 and communications from research groups and national authorities. Forty-three new host plant species of X. fastidiosa, identified through the data extracted from the selected publications, have been added to the database. Those plant species were reported as naturally or artificially infected by subsp. fastidiosa, multiplex, pauca or unknown (i.e. not reported in the publication) subspecies of X. fastidiosa. New information on the tolerant/resistant response of plant species or varieties to X. fastidiosa infection is also reported. No additional data were retrieved for X. taiwanensis. This new version of the database includes no update on the number of Sequence Types (STs) identified so far, which remains unchanged. The overall number of Xylella spp. host plants determined with at least two different detection methods or positive with one method (between: sequencing, pure culture isolation) reaches now 385 plant species, 179 genera and 67 families. Such numbers rise to 638 plant species, 289 genera and 87 families if considered regardless of the detection method applied. The database will be issued twice per year, with the aim to provide information and scientific support to risk assessors, risk managers and researchers dealing with Xylella spp.
Article
Full-text available
Xylella fastidiosa (Xf) is a vascular plant pathogen native to the Americas. In 2013, it was first reported in Europe, implicated in a massive die-off of olive trees in Apulia, Italy. This finding prompted mandatory surveys across Europe, successively revealing that the bacterium was already established in some distant areas of the western Mediterranean. To date, the Balearic Islands (Spain) hold the major known genetic diversity of Xf in Europe. Since October 2016, four sequence types (ST) belonging to the subspecies fastidiosa (ST1), multiplex (ST7, ST81), and pauca (ST80) have been identified infecting 28 host species, including grapevines, almond, olive, and fig trees. ST1 causes Pierce's disease (PD) and together with ST81 are responsible for almond leaf scorch disease (ALSD) in California, from where they were introduced into Mallorca in around 1993, very likely via infected almond scions brought for grafting. To date, almond leaf scorch disease affects over 81% of almond trees and Pierce's disease is widespread in vineyards across Mallorca, although producing on average little economic impact. In this perspective, we present and analyze a large Xf-hosts database accumulated over four years of field surveys, laboratory sample analyses, and research to understand the underlying causes of Xf emergence and spread among crops and wild plants in the Balearic Islands. The impact of Xf on the landscape is discussed.
Article
Full-text available
During recent years; Xylella fastidiosa subsp. pauca (Xfp) has spread in Salento causing relevant damage to the olive groves. Measures to contain the spreading of the pathogen include the monitoring of the areas bordering the so-called "infected" zone and the tree eradication in case of positive detection. In order to provide a control strategy aimed to maintain the tree productivity in the infected areas, we further evaluated the in vitro and in planta mid-term effectiveness of a zinc-copper-citric acid biocomplex. The compound showed an in vitro bactericidal activity and inhibited the biofilm formation in representative strains of X. fastidiosa subspecies, including Xfp isolated in Apulia from olive trees. The field mid-term evaluation of the control strategy assessed by quantitative real-time PCR in 41 trees of two olive groves of the "infected" area revealed a low concentration of Xfp over the seasons upon the regular spraying of the biocomplex over 3 or 4 consecutive years. In particular, the bacterial concentration lowered in July and October with respect to March, after six consecutive treatments. The trend was not affected by the cultivar and it was similar either in the Xfp-sensitive cultivars Ogliarola salentina and Cellina di Nardò or in the Xfp-resistant Leccino. Moreover, the scoring of the number of wilted twigs over the seasons confirmed the trend. The efficacy of the treatment in the management of olive groves subjected to a high pathogen pressure is highlighted by the yielded a good oil production
Article
Full-text available
Xylella fastidiosa subsp. pauca strain De Donno has been recently identified as the causal agent of a severe disease affecting olive trees in a wide area of the Apulia Region (Italy). While insights on the genetics and epidemiology of this virulent strain have been gained, its phenotypic and biological traits remained to be explored. We investigated in vitro behavior of the strain and compare its relevant biological features (growth rate, biofilm formation, cell-cell aggregation, and twitching motility) with those of the type strain Temecula1. The experiments clearly showed that the strain De Donno did not show fringe on the agar plates, produced larger amounts of biofilm and had a more aggregative behavior than the strain Temecula1. Repeated attempts to transform, by natural competence, the strain De Donno failed to produce a GFP-expressing and a knockout mutant for the rpfF gene. Computational prediction allowed us to identify potentially deleterious sequence variations most likely affecting the natural competence and the lack of fringe formation. GFP and rpfF-mutants were successfully obtained by co-electroporation in the presence of an inhibitor of the type I restriction-modification system. The availability of De Donno mutant strains will open for new explorations of its interactions with hosts and insect vectors.
Article
Full-text available
Bacterial phytopathogen Xylella fastidiosa specifically colonizes the plant vascular tissue through a complex process of cell adhesion, biofilm formation, and dispersive movement. Adaptation to the chemical environment of the xylem is essential for bacterial growth and progression of infection. Grapevine xylem sap contains a range of plant secondary metabolites such as phenolics, which fluctuate in response to pathogen infection and plant physiological state. Phenolic compounds are often involved in host-pathogen interactions and influence infection dynamics through signaling activity, antimicrobial properties, and alteration of bacterial phenotypes. The effect of biologically relevant concentrations of phenolic compounds coumaric acid, gallic acid, epicatechin, and resveratrol on growth of X. fastidiosa was assessed in vitro. None of these compounds inhibited bacterial growth, but epicatechin and gallic acid reduced cell-surface adhesion. Cell-cell aggregation decreased with resveratrol treatment, but the other phenolic compounds tested had minimal effect on aggregation. Expression of attachment (xadA) and aggregation (fimA) related genes were altered by presence of the phenolic compounds, consistent with observed phenotypes. All four of the phenolic compounds bound to purified X. fastidiosa lipopolysaccharide (LPS), a major cell-surface component. Information regarding the impact of chemical environment on pathogen colonization in plants is important for understanding the infection process and factors associated with host susceptibility.
Article
Full-text available
Xylella fastidiosa colonizes the xylem of various cultivated and native plants worldwide. Citrus production in Brazil has been seriously affected, and major commercial varieties remain susceptible to Citrus Variegated Chlorosis (CVC). Collective cellular behaviors such as biofilm formation influence virulence and insect transmission of X. fastidiosa. The reference strain 9a5c produces a robust biofilm compared to Fb7 that remains mostly planktonic, and both were isolated from symptomatic citrus trees. This work deepens our understanding of these distinct behaviors at the molecular level, by comparing the cellular and secreted proteomes of these two CVC strains. Out of 1017 identified proteins, 128 showed differential abundance between the two strains. Different protein families were represented such as proteases, hemolysin-like proteins, and lipase/esterases, among others. Here we show that the lipase/esterase LesA is among the most abundant secreted proteins of CVC strains as well, and demonstrate its functionality by complementary activity assays. More severe symptoms were observed in Nicotiana tabacum inoculated with strain Fb7 compared to 9a5c. Our results support that systemic symptom development can be accelerated by strains that invest less in biofilm formation and more in plant colonization. This has potential application in modulating the bacterial-plant interaction and reducing disease severity.
Article
The meadow spittlebug Philaenus spumarius, vector of the bacterium Xylella fastidiosa, relies on vibrational communication to accomplish mating: the female calls to establish a duet with a male. A deeper knowledge of the species’ reproductive biology and behaviour would provide useful information for developing control techniques based on principles of ‘biotremology’, which studies the vibrational behaviour of animals. Playback tests were conducted on single females and male–female pairs of P. spumarius from June to October 2018, and the features of the recorded calling signals were analysed using a wavelet decomposition. Dissections were performed on females to evaluate the relationship between calling activity and ovarioles development. From August onwards, females started to emit calling signals and to develop ovarioles. Female calling activity, duration of their chirps and their responsiveness to mating increased as the season progressed, and they were correlated with ovarioles’ development and presence of mature eggs. Hence, the ovarian maturation represents a key factor in association with the development of the sexual behaviour of P. spumarius females. Conversely, males produced advertisement signals soon after adult eclosion in May, but these signals were not involved in the pair formation process. Mating was achieved only when males produced courtship signals in response to female calling signals and established with them vibrational duets starting from August. Here, we provide new information regarding the P. spumarius’ ethology and hypothesize that potential mating disruption techniques should consider the insect physiology and be applied when both sexes are responsive to mating signals.