Further In Vitro Assessment and Mid-Term Evaluation of Control Strategy of Xylella fastidiosa subsp. pauca in Olive Groves of Salento (Apulia, Italy)

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

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Progress towards Sustainable Control of Xylella fastidiosa subsp. pauca in Olive Groves of Salento (Apulia, Italy)

Pathogens ◽

10.3390/pathogens10060668 ◽

2021 ◽

Vol 10 (6) ◽

pp. 668

Author(s):

Marco Scortichini ◽

Stefania Loreti ◽

Nicoletta Pucci ◽

Valeria Scala ◽

Giuseppe Tatulli ◽

...

Keyword(s):

Control Strategy ◽

Laser Scanning ◽

Vascular System ◽

Xylella Fastidiosa ◽

Low Cost ◽

Interdisciplinary Studies ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Olive Groves

Xylella fastidiosa subsp. pauca is the causal agent of “olive quick decline syndrome” in Salento (Apulia, Italy). On April 2015, we started interdisciplinary studies to provide a sustainable control strategy for this pathogen that threatens the multi-millennial olive agroecosystem of Salento. Confocal laser scanning microscopy and fluorescence quantification showed that a zinc-copper-citric acid biocomplex—Dentamet®—reached the olive xylem tissue either after the spraying of the canopy or injection into the trunk, demonstrating its effective systemicity. The biocomplex showed in vitro bactericidal activity towards all X. fastidiosa subspecies. A mid-term evaluation of the control strategy performed in some olive groves of Salento indicated that this biocomplex significantly reduced both the symptoms and X. f. subsp. pauca cell concentration within the leaves of the local cultivars Ogliarola salentina and Cellina di Nardò. The treated trees started again to yield. A 1H-NMR metabolomic approach revealed, upon the treatments, a consistent increase in malic acid and γ-aminobutyrate for Ogliarola salentina and Cellina di Nardò trees, respectively. A novel endotherapy technique allowed injection of Dentamet® at low pressure directly into the vascular system of the tree and is currently under study for the promotion of resprouting in severely attacked trees. There are currently more than 700 ha of olive groves in Salento where this strategy is being applied to control X. f. subsp. pauca. These results collectively demonstrate an efficient, simple, low-cost, and environmentally sustainable strategy to control this pathogen in Salento.

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The Multi-Millennial Olive Agroecosystem of Salento (Apulia, Italy) Threatened by Xylella Fastidiosa Subsp. Pauca: A Working Possibility of Restoration

Sustainability ◽

10.3390/su12176700 ◽

2020 ◽

Vol 12 (17) ◽

pp. 6700

Author(s):

Marco Scortichini

Keyword(s):

Cell Concentration ◽

Control Strategies ◽

Xylella Fastidiosa ◽

First Record ◽

Interdisciplinary Studies ◽

Insect Vector ◽

Infected Area ◽

Olive Groves ◽

Olive Trees ◽

Spray Treatment

In Salento, the olive agro-ecosystem has lasted more than 4000 years, and represents an invaluable local heritage for landscape, trade, and social traditions. The quarantine bacterium Xylella fastidiosa subsp. pauca was introduced in the area from abroad and has been widely threatening olive groves in the area. The successful eradication of quarantine phytopathogens requires a prompt identification of the causative agent at the new site, a restricted infected area, a highly effective local organization for crop uprooting and biological features of the micro-organism that would guarantee its complete elimination. However, at the time of the first record, these criteria were not met. Interdisciplinary studies showed that a zinc-copper-citric acid biocomplex allowed a consistent reduction of field symptoms and pathogen cell concentration within infected olive trees. In this perspective article, it is briefly described the implementation of control strategies in some olive farms of Salento. The protocol includes spray treatment with the biocomplex during spring and summer, regular pruning of the trees and mowing of soil between February and April to reduce the juvenile of the insect vector(s). Thus far, more than 500 ha have begun to follow this eco-friendly strategy within the “infected” and “containment” areas of Salento.

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Semi-Automatic Method for Early Detection of Xylella fastidiosa in Olive Trees Using UAV Multispectral Imagery and Geostatistical-Discriminant Analysis

Remote Sensing ◽

10.3390/rs13010014 ◽

2020 ◽

Vol 13 (1) ◽

pp. 14

Author(s):

Annamaria Castrignanò ◽

Antonella Belmonte ◽

Ilaria Antelmi ◽

Ruggiero Quarto ◽

Francesco Quarto ◽

...

Keyword(s):

Discriminant Analysis ◽

Early Detection ◽

Plant Pathogens ◽

Early Stage ◽

Xylella Fastidiosa ◽

Mediterranean Countries ◽

Parametric Classification ◽

Aerial Vehicle ◽

Olive Groves ◽

Olive Trees

Xylella fastidiosa subsp. pauca (Xfp) is one of the most dangerous plant pathogens in the world. Identified in 2013 in olive trees in south–eastern Italy, it is spreading to the Mediterranean countries. The bacterium is transmitted by insects that feed on sap, and causes rapid wilting in olive trees. The paper explores the use of Unmanned Aerial Vehicle (UAV) in combination with a multispectral radiometer for early detection of infection. The study was carried out in three olive groves in the Apulia region (Italy) and involved four drone flights from 2017 to 2019. To classify Xfp severity level in olive trees at an early stage, a combined method of geostatistics and discriminant analysis was implemented. The results of cross-validation for the non-parametric classification method were of overall accuracy = 0.69, mean error rate = 0.31, and for the early detection class of accuracy 0.77 and misclassification probability 0.23. The results are promising and encourage the application of UAV technology for the early detection of Xfp infection.

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Neofusicoccum mediterraneum Is Involved in a Twig and Branch Dieback of Olive Trees Observed in Salento (Apulia, Italy)

Pathogens ◽

10.3390/pathogens11010053 ◽

2022 ◽

Vol 11 (1) ◽

pp. 53

Author(s):

Angela Brunetti ◽

Antonio Matere ◽

Valentina Lumia ◽

Vittorio Pasciuta ◽

Valeria Fusco ◽

...

Keyword(s):

Prolonged Exposure ◽

Fungal Species ◽

In Vitro Tests ◽

Wood Discoloration ◽

Olive Groves ◽

Pathogenicity Tests ◽

Olive Quick Decline Syndrome ◽

Olive Trees ◽

Branch Dieback

Olive trees are infected and damaged by Botryosphaeriaceae fungi in various countries. The botryosphaeriaceous fungus Neofusicoccum mediterraneum is highly aggressive and is a major concern for olive groves in Spain and California (USA), where it causes ‘branch and twig dieback’ characterized by wood discoloration, bark canker, and canopy blight. During surveys of olive groves in Apulia (southern Italy), we noticed that—in some areas—trees were heavily affected by severe branch and twig dieback. In addition, chlorosis and the appearance of red-bronze patches on the leaf preceded the wilting of the foliage, with necrotic leaves persisting on the twigs. Given the severity of the manifestation in zones also subject to olive quick decline syndrome (OQDS) caused by Xylella fastidiosa subsp. pauca, we investigated the etiology and provide indications for differentiating the symptoms from OQDS. Isolation from diseased wood samples revealed a mycete, which was morphologically and molecularly identified as N. mediterraneum. The pathogenicity tests clearly showed that this fungus is able to cause the natural symptoms. Therefore, also considering the low number of tested samples, N. mediterraneum is a potential causal agent of the observed disease. Specifically, inoculation of the twigs caused complete wilting in two to three weeks, while inoculation at the base of the stem caused severe girdling wedge-shaped cankers. The growth rate of the fungus in in vitro tests was progressively higher from 10 to 30 °C, failing to grow at higher temperatures, but keeping its viability even after prolonged exposure at 50 °C. The capacity of the isolate to produce catenulate chlamydospores, which is novel for the species, highlights the possibility of a new morphological strain within N. mediterraneum. Further investigations are ongoing to verify whether additional fungal species are involved in this symptomatology.

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Expression of Pathogenicity-Related Genes of Xylella fastidiosa In Vitro and In Planta

Current Microbiology ◽

10.1007/s00284-004-4447-8 ◽

2005 ◽

Vol 50 (4) ◽

pp. 223-228 ◽

Cited By ~ 28

Author(s):

Alessandra A. de Souza ◽

Marco A. Takita ◽

Eridan O. Pereira ◽

Helvécio D. Coletta-Filho ◽

Marcos A. Machado

Keyword(s):

Xylella Fastidiosa ◽

In Planta

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Copper Supplementation in Watering Solution Reaches the Xylem But Does Not Protect Tobacco Plants Against Xylella fastidiosa Infection

Plant Disease ◽

10.1094/pdis-08-19-1748-re ◽

2020 ◽

Vol 104 (3) ◽

pp. 724-730 ◽

Cited By ~ 2

Author(s):

Qing Ge ◽

Paul A. Cobine ◽

Leonardo De La Fuente

Keyword(s):

Biofilm Formation ◽

Inductively Coupled Plasma ◽

Tap Water ◽

Xylella Fastidiosa ◽

In Vitro Study ◽

Optical Emission ◽

In Planta ◽

Inductively Coupled ◽

High Concentrations

Xylella fastidiosa is a xylem-limited plant pathogenic bacterium that causes disease in many crops worldwide. Copper (Cu) is an antimicrobial agent widely used on X. fastidiosa hosts to control other diseases. Although the effects of Cu for control of foliar pathogens are well known, it is less studied on xylem-colonizing pathogens. Previous results from our group showed that low concentrations of CuSO4 increased biofilm formation, whereas high concentrations inhibited biofilm formation and growth in vitro. In this study, we conducted in planta experiments to determine the influence of Cu in X. fastidiosa infection using tobacco as a model. X. fastidiosa-infected and noninfected plants were watered with tap water or with water supplemented with 4 mM or 8 mM of CuSO4. Symptom progression was assessed, and sap and leaf ionome analysis was performed by inductively coupled plasma with optical emission spectroscopy. Cu uptake was confirmed by increased concentrations of Cu in the sap of plants treated with CuSO4-amended water. Leaf scorch symptoms in Cu-supplemented plants showed a trend toward more severe at later time points. Quantification of total and viable X. fastidiosa in planta indicated that CuSO4-amended treatments did not inhibit but slightly increased the growth of X. fastidiosa. Cu in sap was in the range of concentrations that promote X. fastidiosa biofilm formation according to our previous in vitro study. Based on these results, we proposed that the plant Cu homeostasis machinery controls the level of Cu in the xylem, preventing it from becoming elevated to a level that would lead to bacterial inhibition.

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Detection and Visualization of an Exopolysaccharide Produced by Xylella fastidiosa In Vitro and In Planta

Applied and Environmental Microbiology ◽

10.1128/aem.00895-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7252-7258 ◽

Cited By ~ 33

Author(s):

M. Caroline Roper ◽

L. Carl Greve ◽

John M. Labavitch ◽

Bruce C. Kirkpatrick

Keyword(s):

Xanthan Gum ◽

Xanthomonas Campestris ◽

Polyclonal Antibodies ◽

Xylella Fastidiosa ◽

Enzyme Linked Immunosorbent Assay ◽

In Planta ◽

Mannose Residue ◽

Pantoea Stewartii ◽

Vascular Occlusions

ABSTRACT Many phytopathogenic bacteria, such as Ralstonia solanacearum, Pantoea stewartii, and Xanthomonas campestris, produce exopolysaccharides (EPSs) that aid in virulence, colonization, and survival. EPS can also contribute to host xylem vessel blockage. The genome of Xylella fastidiosa, the causal agent of Pierce's disease (PD) of grapevine, contains an operon that is strikingly similar to the X. campestris gum operon, which is responsible for the production of xanthan gum. Based on this information, it has been hypothesized that X. fastidiosa is capable of producing an EPS similar in structure and composition to xanthan gum but lacking the terminal mannose residue. In this study, we raised polyclonal antibodies against a modified xanthan gum polymer similar to the predicted X. fastidiosa EPS polymer. We used enzyme-linked immunosorbent assay to quantify production of EPS from X. fastidiosa cells grown in vitro and immunolocalization microscopy to examine the distribution of X. fastidiosa EPS in biofilms formed in vitro and in planta and assessed the contribution of X. fastidiosa EPS to the vascular occlusions seen in PD-infected grapevines.

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Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection

Phytopathology ◽

10.1094/phyto-02-16-0097-r ◽

2016 ◽

Vol 106 (8) ◽

pp. 928-936 ◽

Cited By ~ 6

Author(s):

Lindsey P. Burbank ◽

Drake C. Stenger

Keyword(s):

Copy Number ◽

Xylella Fastidiosa ◽

Expression Vectors ◽

Broad Host Range ◽

In Planta ◽

Plasmid Vectors ◽

Antibiotic Selection ◽

Shuttle Vectors ◽

Range Vector

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

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How Ecosystem Services Can Strengthen the Regeneration Policies for Monumental Olive Groves Destroyed by Xylella fastidiosa Bacterium in a Peri-Urban Area

Sustainability ◽

10.3390/su13168778 ◽

2021 ◽

Vol 13 (16) ◽

pp. 8778

Author(s):

Teodoro Semeraro ◽

Elisa Gatto ◽

Riccardo Buccolieri ◽

Valentina Catanzaro ◽

Luigi De Bellis ◽

...

Keyword(s):

Land Use ◽

Ecosystem Services ◽

Land Cover ◽

Cultural Heritage ◽

Urban Area ◽

Urban Areas ◽

Xylella Fastidiosa ◽

Trade Offs ◽

Olive Groves ◽

Olive Trees

The Apulian Region (Italy) is a socio-ecological system shaped by the millennial co-evolution between human actions and ecological processes. It is characterized by monumental olive groves protected from Regional Law 14/2007 for the cultural value of the landscape, currently threatened by the spread of a devastating phytopathogen, the bacteria Xylella fastidiosa. The aim of this paper is to apply landscape resilience analysis focusing on ecosystem services to understand the potential effects and trade-offs of regeneration policies in a peri-urban area characterized by monumental olive groves land cover. The study involved land-cover and land-use analysis, supported by a survey on the inhabitants and an ecosystem services analysis. The results showed a mismatch between the agroecosystem and the social and economic use linked to leisure or hospitality. The study area was defined as a peri-urban landscape characterized by tourist use. From the interviews of the users, the cultural heritage of olive groves seems linked to the presence of olive trees like a status quo of the landscape and olive oil productions. The culture aspect could thus be preserved by changing the type of olive trees. In addition, the analysis showed that the microclimate could be preserved and enhanced in terms of air temperature and thermal comfort, by replacing the olive trees with varieties resistant to Xylella, such as cv. Leccino. Therefore, regeneration policies that promote replacing dead olive groves with new olive trees could be efficient to stimulate social components of the landscape and improve the resilience of ecosystem services in peri-urban areas in the interest of the cultural heritage of the users and benefits that they provide. An ecosystem services analysis at a local scale could be a strategy for an integrated regenerate approach between land-use and land-cover with social, ecological, and economic evolutions vision orientated to a sustainable and desirable future.

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Chromosome-Based Genetic Complementation System for Xylella fastidiosa

Applied and Environmental Microbiology ◽

10.1128/aem.00024-09 ◽

2009 ◽

Vol 75 (6) ◽

pp. 1679-1687 ◽

Cited By ~ 36

Author(s):

Ayumi Matsumoto ◽

Glenn M. Young ◽

Michele M. Igo

Keyword(s):

Dna Sequences ◽

Xylella Fastidiosa ◽

Cell Physiology ◽

Multiple Cloning Site ◽

Wild Type ◽

In Planta ◽

Catalase Peroxidase ◽

The Stability

ABSTRACT Xylella fastidiosa is a xylem-limited, gram-negative bacterium that causes Pierce's disease of grapevine. Here, we describe the construction of four vectors that facilitate the insertion of genes into a neutral site (NS1) in the X. fastidiosa chromosome. These vectors carry a colE1-like (pMB1) replicon and DNA sequences from NS1 flanking a multiple-cloning site and a resistance marker for one of the following antibiotics: chloramphenicol, erythromycin, gentamicin, or kanamycin. In X. fastidiosa, vectors with colE1-like (pMB1) replicons have been found to result primarily in the recovery of double recombinants rather than single recombinants. Thus, the ease of obtaining double recombinants and the stability of the resulting insertions at NS1 in the absence of selective pressure are the major advantages of this system. Based on in vitro and in planta characterizations, strains carrying insertions within NS1 are indistinguishable from wild-type X. fastidiosa in terms of growth rate, biofilm formation, and pathogenicity. To illustrate the usefulness of this system for complementation analysis, we constructed a strain carrying a mutation in the X. fastidiosa cpeB gene, which is predicted to encode a catalase/peroxidase, and showed that the sensitivity of this mutant to hydrogen peroxide could be overcome by the introduction of a wild-type copy of cpeB at NS1. Thus, this chromosome-based complementation system provides a valuable genetic tool for investigating the role of specific genes in X. fastidiosa cell physiology and virulence.

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