pathogens

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

Marco Scortichini 1,*, Stefania Loreti 2 , Nicoletta Pucci 2 , Valeria Scala 2 , Giuseppe Tatulli 2, Dimitri Verweire 3, Michael Oehl 3, Urs Widmer 3, Josep Massana Codina 3 , Peter Hertl 3, Gianluigi Cesari 4, Monica De Caroli 5 , Federica Angilè 5, Danilo Migoni 5, Laura Del Coco 5 , Chiara Roberta Girelli 5 , Giuseppe Dalessandro 5 and Francesco Paolo Fanizzi 5

1 Research Centre for Olive, Fruit Trees and Citrus Crops, Council for Agricultural Research and Economics (CREA), 00134 Roma, Italy 2 Research Centre for Plant Protection and Certification, Council for Agricultural Research and Economics (CREA), 00156 Roma, Italy; [email protected] (S.L.); [email protected] (N.P.); [email protected] (V.S.); [email protected] (G.T.) 3 Invaio Sciences, Cambridge, MA 02138, USA; [email protected] (D.V.); [email protected] (M.O.); [email protected] (U.W.); [email protected] (J.M.C.); [email protected] (P.H.) 4 Agrifound Italia, 50122 Firenze, Italy; [email protected] 5 Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Monteroni-Lecce, Italy; [email protected] (M.D.C.); [email protected] (F.A.); [email protected] (D.M.); [email protected] (L.D.C.); [email protected] (C.R.G.); [email protected] (G.D.); [email protected] (F.P.F.) * Correspondence: [email protected]



 Abstract: Xylella fastidiosa subsp. pauca is the causal agent of “olive quick decline syndrome” in Citation: Scortichini, M.; Loreti, S.; Salento (Apulia, Italy). On April 2015, we started interdisciplinary studies to provide a sustainable Pucci, N.; Scala, V.; Tatulli, G.; control strategy for this pathogen that threatens the multi-millennial olive agroecosystem of Salento. Verweire, D.; Oehl, M.; Widmer, U.; Confocal laser scanning microscopy and fluorescence quantification showed that a zinc-copper-citric Codina, J.M.; Hertl, P.; et al. Progress acid biocomplex—Dentamet®—reached the olive xylem tissue either after the spraying of the canopy towards Sustainable Control of Xylella or injection into the trunk, demonstrating its effective systemicity. The biocomplex showed in vitro fastidiosa subsp. pauca in Olive Groves of Salento (Apulia, Italy). Pathogens bactericidal activity towards all X. fastidiosa subspecies. A mid-term evaluation of the control strategy 2021, 10, 668. https://doi.org/ performed in some olive groves of Salento indicated that this biocomplex significantly reduced 10.3390/pathogens10060668 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 Academic Editor: Hon Ho 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 Received: 28 April 2021 technique allowed injection of Dentamet® at low pressure directly into the vascular system of the Accepted: 26 May 2021 tree and is currently under study for the promotion of resprouting in severely attacked trees. There Published: 29 May 2021 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 Publisher’s Note: MDPI stays neutral environmentally sustainable strategy to control this pathogen in Salento. with regard to jurisdictional claims in published maps and institutional affil- Keywords: olive quick decline syndrome; real-time PCR; NMR metabolomic; endotherapy; sustain- iations. able development goals of the United Nations

Copyright: © 2021 by the authors. 1. The Phytosanitary Emergencies Licensee MDPI, Basel, Switzerland. This article is an open access article During recent years, the increase of global plant exports for agriculture and forestry distributed under the terms and has dramatically augmented the probability for phytopathogens to rapidly reach new conditions of the Creative Commons countries and, consequently, to colonize and infect new crops and/or the same crop Attribution (CC BY) license (https:// cultivated in another continent [1]. It should be said, however, that the introduction of creativecommons.org/licenses/by/ pathogens and pests in a new area has also been observed in the past. For Puccinia striiformis 4.0/). f. sp. tritici, the causal agent of wheat yellow rust, it was established that the introduction

Pathogens 2021, 10, 668. https://doi.org/10.3390/pathogens10060668 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 668 2 of 22

of new populations of the pathogen into areas of wheat cultivation appear to be linked to commerce and travel over the last two centuries [2]. Pathogens can be hosted by asymptomatic potted plants, plant parts, seeds, bulbs, and timber as latent but viable cells of the pathogen that can start new infections when favorable conditions occur. The unintended introduction of new pests and pathogens in a new country poses serious risks for both cultivated crops and natural ecosystems [3]. Recent examples of newly introduced pathogens which caused severe damage to natural ecosystems include Chryphonectria parasitica in chestnut in southeastern Europe [4], and Phytopthora ramorum in oak species in the U.S.A. and in larch in the United Kingdom and Ireland [5,6]. Examples in cultivated crops include kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae introduced worldwide from eastern Asia in all major areas of Actinidia chinensis cultivation [7], and the “olive quick decline syndrome” (OQDS), caused by Xylella fastidiosa subsp. pauca introduced from Central America in Salento (Apulia, Italy) [8,9]. Dispersal of novel pathogens within an area could also be caused by many human activities not directly linked with the agricultural trade [10]. Within this context, countries with higher economic activity and population density tend to spread a greater number of biological threats [11]. Once introduced into a new area, the phytopathogens can become a permanent part of the new environment(s) depending on a series of factors such as the number of introduction events, the transmission rate of the pathogen, the density and spatial variation of suscep- tible host(s), the climatic conditions, and the synchronicity between host susceptibility and pathogen life cycle [12]. The climatic conditions and the complex organization of agricultural production and trade prevailing in the countries facing the Mediterranean Basin appear particularly favorable for the introduction and rapid establishment of ex- otic phytopathogens. This area is characterized by mild and wet winters that favor the transmission rate of pathogens and by a very rich array of cultivated and forest crops that feed the global circulation of propagative plant material [12]. The intense year-round touristic activities in this area also favor the unintended displacement and introduction of pests and pathogens from other continents [12]. Upon their introduction into a new area, the emergent pathogen should be eradicated if possible (e.g., through prompt pathogen detection, restricting the area of infection, and organized human activities) or it should be controlled according to known or new strategies to limit the disease severity and its further spread [13].

2. The Concept of Pathogen Control in Plant Pathology and Xylella Fastidiosa The main aim in plant disease management is to reduce the economic impact of pathogens on cultivated crops [14] through control strategies that can vary according to the cropping system, the climatic features of the area, and the agronomic techniques applied to the crop [15]. Controlling a plant pathogenic bacterium does not necessarily imply its elimination from the crop but rather a significant reduction of its inoculum size that allows the crop to regularly yield [13]. In contrast, control of X. fastidiosa implies its elimination from the diseased plant [16], due to the quarantine status of the pathogen for Europe and the concern for its possible spread in other territories of the continent. This is a crucial and difficult task, especially when this endophytic pathogen is well-established in an area long before its first detection, as in the case of the OQDS in Salento. Eradicating X. fastidiosa is made more difficult by the bacterium’s ability to colonize many wild plant species and to be effectively spread in a territory by insect vectors. Cultivars that have retained resistane to X. f. subsp. pauca, such as Leccino, have limited multiplication of the pathogen within xylem tissue but do not eliminate the bacterium from the plant [17]; consequently, growing such cultivars does not achieve the goal of eliminating X. fastidiosa from the area. It would therefore appear that this pathogen, when not promptly detected in a new area, has a very high probability of becoming established there and destroying the cultivated crop in few years. In contrast, satisfactory reductions Pathogens 2021, 10, 668 3 of 22

of grapevine Pierce’s disease caused by X. f. subsp. fastidiosa were recently obtained with a weakly virulent strain of the pathogen [18] or with a novel bacterial biocontrol agent [19]. From this “control” perspective, more efforts should be made to identify strategies that significantly reduce the bacterial inoculum size within the plant across the seasons. This approach, coupled with an effective control of the insect vector(s), could allow to limit both the severity and the spread of X. fastidiosa in a territory, allowing crops to coexist with the pathogen similar to other bacterial diseases.

3. Xylella Fastidiosa subsp. Pauca in Apulia, Italy The first record of an association of X. fastidiosa with olive groves showing twig and branch wilting in the Lecce province (Salento, Apulia) was reported on October 2013 [20]. The bacterium was isolated in cultures and characterized as belonging to the subspecies pauca, and the common name of “olive quick decline syndrome” was proposed for the disease [21]. Th main symptoms include leaf, twig and branch wilting, followed by the death of the tree. The local cultivars Ogliarola salentina and Cellina di Nardò are very sensitive to the disease. At the time of the first record, the disease was already spread over 8000 to 10,000 hectares of the Salento territory, corresponding to about 800,000 to 1,000,000 olive trees [22]. At that time, X. fastidiosa was added to the A1 list of quarantine pathogens for the European and Mediterranean Plant Protection Organization (EPPO), and, consequently, eradication measures had to be implemented, even though previous experience and the relatively widespread occurrence of the pathogen in the area made a successful outcome doubtful [23]. After an initial attempt to eradicate trees, an area of Salento was officially designed as “infected” and additional “containment” and “buffer” areas were established further north to monitor any occurrence of the pathogen and to prevent its spread within the region (decision of the European Union, 2016). Notwithstanding this, during the following years, X. f. subsp. pauca was reported in other olive groves of Salento in the Brindisi and Pathogens 2021, 10, x FOR PEER REVIEW 4 of 22 Taranto provinces [24], so the extension and placement of “containment” and “buffer” areas changed during the years (decision of the European Union, 2020) (Figure1).

Figure 1. DemarcationFigure 1. of Demarcation the “infected” of the (southern “infected” areas), (southern “containment” areas), “containment” (yellow) and (yellow) “buffer” and “buffer” (blue) areas of Apulia (blue) areas of Apulia according to the "Aggiornamento delle aree delimitate alla Xylella fastidiosa according to the “Aggiornamento delle aree delimitate alla Xylella fastidiosa sottospecie pauca ST53”, based on the decision of sottospecie pauca ST53", based on the decision of the European Union 2020/1201 and on the decision the European Union 2020/1201 and on the decision of the Apulia region n◦ 548/2020. The “containment” and “buffer” of the Apulia region n° 548/2020. The “containment” and “buffer” areas are being monitored to areas are being monitoredreveal the occurrence to reveal the of new occurrence olive an ofd other new oliveplant and species other infected plant speciesby Xylella infected fastidiosa by subsp.Xylella pauca fastidiosa. subsp. pauca. ReproducedReproduced from Regione from Regione Apulia website: Apulia website: www.emergenzaxylella.it www.emergenzaxylella.it.

4. A Control Strategy to Preserve a Multi-millennial Agroecosystem The centuries-old practices employed in the olive groves of Salento, such as exten- sive tree management of the local cultivars and the traditional way of olive picking, characterize the whole area as a typical Mediterranean olive agroecosystem that also represents a remarkable historical, cultural, and landscaped heritage [26] (Figure 2). The local cultivars—Ogliarola salentina and Cellina di Nardò—are among the richest olive cultivars in polyphenol content and can yield an oil with a very high nutritional value [27,28]. Due to their sensitivity to the disease, the risk of losing these cultivars to disease is very high. These features of the territory prompted us to find possible field strategies that could limit both the severity and spread of the OQDS in Apulia, including identify- ing treatments that offer a sustainable approach to the problem and might potentially effectively limit the X. f. subsp. pauca inoculum within the plant. The preservation of such agro-ecosystem fulfills the Sustainable Development Goals (SDG) of the United Nations, namely SDG 15: “Protect, restore and promote sustainable use of terrestrial ecosystems, sustainably manage forests, combat desertification, and halt and reverse land degrada- tion and halt biodiversity loss” (https://sdgs.un.org/goals/goal15, accessed on: 24 April 2021).

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Currently, the pathogen is included into the A2 list of quarantine pathogens for EPPO, and a recent analysis has estimated that in Salento, about 6,500,000 olive trees are infected by the bacterium [25]. The monitoring plan to detect infected olive trees in the “containment” and “buffer” areas is under way, and it is carried out by the institutions of the Apulia region.

4. A Control Strategy to Preserve a Multi-millennial Agroecosystem The centuries-old practices employed in the olive groves of Salento, such as exten- sive tree management of the local cultivars and the traditional way of olive picking, characterize the whole area as a typical Mediterranean olive agroecosystem that also repre- sents a remarkable historical, cultural, and landscaped heritage [26] (Figure2). The local cultivars—Ogliarola salentina and Cellina di Nardò—are among the richest olive cultivars in polyphenol content and can yield an oil with a very high nutritional value [27,28]. Due to their sensitivity to the disease, the risk of losing these cultivars to disease is very high. These features of the territory prompted us to find possible field strategies that could limit both the severity and spread of the OQDS in Apulia, including identifying treatments that offer a sustainable approach to the problem and might potentially effectively limit the X. f. subsp. pauca inoculum within the plant. The preservation of such agro-ecosystem fulfills the Sustainable Development Goals (SDG) of the United Nations, namely SDG 15: “Protect, restore and promote sustainable use of terrestrial ecosystems, sustainably manage forests, Pathogens 2021, 10, x FOR PEER REVIEW 5 of 22 combat desertification, and halt and reverse land degradation and halt biodiversity loss” (https://sdgs.un.org/goals/goal15, accessed on: 24 April 2021).

Figure 2. A continuum ofFigure olive trees2. A that continuum extend over of kilometers olive trees characterizes that extend the multi-millennialover kilometers olive characterizes agro-ecosystem the of mul- Salento (Apulia, Italy). ti-millennial olive agro-ecosystem of Salento (Apulia, Italy).

5. The5. The Basic Basic Knowledge Knowledge and and Criteria Criteria forfor Choosing Choosing an an Effective Effective Compound Compound In Inorder order to to select select a a candidate candidate formulationformulation for for establishing establishing an effectivean effective field field control control strategy,strategy, an an understanding understanding of of bacterial pathosystems pathosystems is requiredis required for identifyingfor identifying potential potential targets in the pathogen as well as possible seasonal timings for deployment [13]. A review targets in the pathogen as well as possible seasonal timings for deployment [13]. A re- of the scientific literature concerning the effectiveness of substances in inhibiting the in vitro viewgrowth of the and scientific biofilm formationliterature ofconcerningX. fastidiosa therevealed effectiveness that the ionomic of substances content in of inhibiting the plant the in vitroplays growth a role in and the modulating biofilm formation the virulence of X. of fastidiosa the pathogen. revealed Zinc that and the copper ionomic ions, atcontent cer- of thetain plant concentrations, plays a role have in the been modulating reported to the act asvirulence effective inhibitorsof the pathogen. of bacterium’s Zinc vitality.and copper ions, at certain concentrations, have been reported to act as effective inhibitors of bacte- rium’s vitality. Copper at > 0.2 mM and zinc at > 0.25 mM inhibit biofilm formation [29], whereas a sublethal concentration of zinc promotes biofilm development [30]. Zinc de- toxification by the bacterium within xylematic tissue of the host plant is required for full virulence, suggesting that an additional supplement of zinc ion could inhibit xylem pathogen colonization [31]. Thus, manipulation of zinc and/or copper in the plant is a potential disease management strategy [31]. X. fastidiosa is characterized by an endo- phytic lifestyle completely within the xylem tissue. This means that the bactericidal copper compounds commonly used in topical applications to control other bacterial plant pathogens cannot reach the pathogen cells since these treatments act mainly on the plant’s surfaces and are not systemic, and therefore are not used in treating diseases caused by X. fastidiosa. Formulations for treating a pathogen limited to the xylem must be reliably systemic. Kirkpatrick et al. [32], referring to Pierce’s disease of grapevine, ar- gued, “if methods could be developed to effectively deliver prophylactic or therapeutic bactericides into grapevine, this could provide a comparatively straightforward solution to a very complex disease problem”. Thus, a suitable compound for controlling X. fastidiosa must be bactericidal and systemic, and ideally environmentally friendly [15] and sustainable for the olive agroe- cosystem. Moreover, its cost should preferably be low and its handling should be easy. Based on these criteria, we have chosen the internationally patented biofertilizer Den- tamet® for further evaluation. Dentamet® contains a mixture of zinc (4% w/w) and cop- per (2% w/w) that is complexed with hydracids of citric acid through a fermentation process similar to those occurring naturally in some soil fungi. Due to its low environ- mental impact, this biofertilizer is also allowed for organic farming. We have i) per- formed in vitro assays to test the bactericidal activity of Dentamet® towards all X. fastid- iosa subspecies, ii) ascertained the systemicity of Dentamet® within the olive xylem after

Pathogens 2021, 10, 668 5 of 22

Copper at > 0.2 mM and zinc at > 0.25 mM inhibit biofilm formation [29], whereas a sub- lethal concentration of zinc promotes biofilm development [30]. Zinc detoxification by the bacterium within xylematic tissue of the host plant is required for full virulence, suggesting that an additional supplement of zinc ion could inhibit xylem pathogen colonization [31]. Thus, manipulation of zinc and/or copper in the plant is a potential disease management strategy [31]. X. fastidiosa is characterized by an endophytic lifestyle completely within the xylem tissue. This means that the bactericidal copper compounds commonly used in topical applications to control other bacterial plant pathogens cannot reach the pathogen cells since these treatments act mainly on the plant’s surfaces and are not systemic, and therefore are not used in treating diseases caused by X. fastidiosa. Formulations for treating a pathogen limited to the xylem must be reliably systemic. Kirkpatrick et al. [32], referring to Pierce’s disease of grapevine, argued, “if methods could be developed to effectively deliver prophylactic or therapeutic bactericides into grapevine, this could provide a comparatively straightforward solution to a very complex disease problem”. Thus, a suitable compound for controlling X. fastidiosa must be bactericidal and systemic, and ideally environmentally friendly [15] and sustainable for the olive agroe- cosystem. Moreover, its cost should preferably be low and its handling should be easy. Based on these criteria, we have chosen the internationally patented biofertilizer Dentamet® for further evaluation. Dentamet® contains a mixture of zinc (4% w/w) and copper (2% w/w) that is complexed with hydracids of citric acid through a fermentation process similar to those occurring naturally in some soil fungi. Due to its low environmental impact, this biofertilizer is also allowed for organic farming. We have (i) performed in vitro assays to test the bactericidal activity of Dentamet® towards all X. fastidiosa subspecies, (ii) ascer- tained the systemicity of Dentamet® within the olive xylem after foliar spraying or injection, (iii) measured the effective release of zinc and copper ions within the olive xylem, (iv) ascertained the capability of Dentamet® applied by foliar spray to reduce OQDS symptoms in field trials in Salento olive orchards, (v) evaluated X. fastidiosa subsp. pauca seasonal cell concentrations in treated olive foliage by quantitative real-time PCR, (vi) ascertained the absence of copper and zinc in oil produced from treated olive trees, (vii) and considered the possibility of inciting twig resprouting in very severely damaged trees through Dentamet® trunk injection.

6. Interdisciplinary Studies to Set Up a Sustainable Control Strategy Dentamet® biofertilizer showed bactericidal activity towards all the X. f. subsp. fastidiosa, multiplex and pauca strains tested, including the one isolated from olive trees showing OQDS symptoms in Apulia [33,34]. The antibacterial activity was ascertained both in broth tubes and on a bacterial culture substrate. The absence/reduction of growth was ascertained through real-time PCR. The minimum bactericidal concentration (MBC) for the biofertilizer was 400 mg/L and at 200 mg/L for zinc and copper, respectively (Figure3). Dentamet ® also significantly reduced the biofilm formation in all strains tested (Figure4). These data indicated that the biofertilizer can reduce both the pathogen vessel colonization (planktonic phase) and the biofilming phase, and justified further testing in the field. Confocal laser scanning microscopy and fluorescence quantification confirmed that Dentamet® reached the olive xylem tissue either after the spraying of the canopy or injection into the trunk, demonstrating its effective systemicity. Fluorescent staining of the xylem tissue of the leaf, leaf petiole and of two- and five-year-old twigs clearly showed the migration of the biofertilizer from the entry point (Figure5). The same tissue samples were also assessed for zinc and copper release through coupled plasma atomic emission spectroscopy, and the ions were effectively detected and quantified in the olive tissue [33]. These data indicated that the biofertilizer reaches the xylem tissue where X. f. subsp. pauca lives and multiplies. Pathogens 2021, 10, x FOR PEER REVIEW 6 of 22

foliar spraying or injection, iii) measured the effective release of zinc and copper ions within the olive xylem, iv) ascertained the capability of Dentamet® applied by foliar spray to reduce OQDS symptoms in field trials in Salento olive orchards, v) evaluated X. fastidiosa subsp. pauca seasonal cell concentrations in treated olive foliage by quantitative real-time PCR, vi) ascertained the absence of copper and zinc in oil produced from treated olive trees, vii) and considered the possibility of inciting twig resprouting in very severely damaged trees through Dentamet® trunk injection.

6. Interdisciplinary Studies to Set Up a Sustainable Control Strategy Dentamet® biofertilizer showed bactericidal activity towards all the X. f. subsp. fas- tidiosa, multiplex and pauca strains tested, including the one isolated from olive trees showing OQDS symptoms in Apulia [33.34]. The antibacterial activity was ascertained both in broth tubes and on a bacterial culture substrate. The absence/reduction of growth was ascertained through real-time PCR. The minimum bactericidal concentration (MBC) for the biofertilizer was 400 mg/L and at 200 mg/L for zinc and copper, respectively (Figure 3). Dentamet® also significantly reduced the biofilm formation in all strains tested (Figure 4). These data indicated that the biofertilizer can reduce both the pathogen vessel colonization (planktonic phase) and the biofilming phase, and justified further testing in the field. Confocal laser scanning microscopy and fluorescence quantification confirmed that Dentamet® reached the olive xylem tissue either after the spraying of the canopy or injection into the trunk, demonstrating its effective systemicity. Fluorescent staining of the xylem tissue of the leaf, leaf petiole and of two- and five-year-old twigs clearly showed the migration of the biofertilizer from the entry point (Figure 5). The same tissue samples were also assessed for zinc and copper release through coupled plasma

Pathogens 2021, 10, 668 atomic emission spectroscopy, and the ions were effectively detected and quantified6 of 22in the olive tissue [33]. These data indicated that the biofertilizer reaches the xylem tissue where X. f. subsp. pauca lives and multiplies.

Figure 3. Bactericidal evaluationFigure of3. XylellaBactericidal fastidiosa evaluationsubsp. paucaof Xylellastrain fastidiosa De Donno subsp. CFBP8402 pauca strain (A), X.Def. Donnosubsp. CFBP8402fastidiosa (A), X. f. subsp. fastidiosa strain Temecula1 (B), and X. f. subsp. multiplex strain CFBP 8416 (C). strain Temecula1 (B), and X. f. subsp. multiplex strain CFBP 8416 (C). Aliquots of 10 µL and 10-fold dilutions from 107 to Aliquots of 10 μL and 10-fold dilutions from 107 to 103 CFU mL−1 of each X. fastidiosa subspe- 103 CFU mL−1 of each X. fastidiosa subspecies were spotted on PD2 medium agar plates supplemented or not with 1:10, cies were spotted on PD2 medium agar plates supplemented or not with 1:10, 1:50 and 1:100 ® ◦ X. fastidiosa 1:50 and 1:100 Dentamet dilutionsDentamet® and dilutions incubated and at incuba 28 C.ted Ctr at (control):28 °C. Ctr (control): X.cultures fastidiosa grown cultures on grown PD2 medium on PD2 ® Dentamet -free. Reproducedmedium from [ 34Dentamet®-free.]. Reproduced from [34]. For the field trials, three representative olive farms all located in Salento (Lecce For the field trials, three representative olive farms all located in Salento (Lecce province) in municipalities characterized by a severe occurrence of OQDS were selected, province) in municipalities characterized by a severe occurrence of OQDS were selected, namely Veglie, Cannole and Galatone. The occurrence of X. f. subsp. pauca was ascertained on all farms before starting the trial. All olive groves were managed according to the traditional cultivation technique usually carried out in Salento: no irrigation, no regular pruning or soil fertilization, control of the main pests, free vase training system, and ample spacing between the trees. Dentamet® was applied by foliar spray, i.e., by nebulization on the olive crown using an atomizer, at 3.9 kg/ha, once per month from April to September. An initial three-year trial was established beginning April 2015 at Veglie to assess the potential of Dentamet® in reducing the impact of X. f. subsp. pauca to the local olive cultivars Ogliarola salentina and Cellina di Nardò. Subsequent trials performed at Cannole and Galatone during 2019 represented a mid-term evaluation of the pathogen control effectiveness, having started the regular application of Dentamet® in those olive groves during the previous four and three years, respectively. A significant reduction of both field symptoms (i.e., twig wilting) and pathogen concentration within the leaves were observed at Veglie. It worth noting that during the study, apart from X. f. subsp. pauca infection, the trees faced some adverse climatic events such as frost in January 2017 and a severe heat wave during summer 2017 [33,35]. It was observed that at least 50 to 60% of the canopy should be present in the tree for satisfactory entry of Dentamet® into the xylem tissue. No zinc and copper residues were found within the oil obtained from trees that received the biofertilizer over three years. Pathogens 2021, 10, x FOR PEER REVIEW 7 of 22

namely Veglie, Cannole and Galatone. The occurrence of X. f. subsp. pauca was ascer- tained on all farms before starting the trial. All olive groves were managed according to the traditional cultivation technique usually carried out in Salento: no irrigation, no reg- ular pruning or soil fertilization, control of the main pests, free vase training system, and Pathogens 2021, 10, 668 ample spacing between the trees. Dentamet® was applied by foliar spray, i.e., by nebu-7 of 22 lization on the olive crown using an atomizer, at 3.9 kg/ha, once per month from April to September.

FigureFigure 4. 4. BiofilmBiofilm assay assay 30 30 dpi dpi of of XylellaXylella fastidiosa fastidiosa subspsubsp.. paucapauca (De(De Donno_CFBP8402) Donno_CFBP8402) (Xfp) (Xfp) ( (AA),), and and 1515 dpi dpi of of X.X. fastidiosa fastidiosa subsp.subsp. fastidiosafastidiosa (Temecula1)(Temecula1) (Xff) (Xff) (B (B) )and and XX fastidiosa fastidiosa subsp.subsp. multiplexmultiplex (CFBP(CFBP 8416)8416) (Xfm) (Xfm) ( (CC).). The The ordinate ordinate axes axes report report % % of of biofilm biofilm formation, formation, the the abscis abscissasa axes axes the the Dentamet Dentamet® ® dilutions.dilutions. Values Values are are means means ±± SDSD of of three three independent independent biological biological replicates replicates (n (=n 7).= 7).A statistically A statistically significantsignificant difference difference was was obtained obtained between between Xfp, Xfp, Xff Xff and and Xfm Xfm PD2 PD2 cultures cultures (untreated (untreated control) control) and and the same subspecies added with Dentamet® dilutions (normalized with absorbance obtained by the same subspecies added with Dentamet® dilutions (normalized with absorbance obtained by tubes without bacteria), according to one-way ANOVA, Dunnett’s test (**** p ≤ 0.0001 vs. Control). tubes without bacteria), according to one-way ANOVA, Dunnett’s test (**** p ≤ 0.0001 vs. Control). Reproduced from [34]. Reproduced from [34]. An initial three-year trial was established beginning April 2015 at Veglie to assess Finally, extensive twig resprouting in severely attacked trees was observed upon the potential of Dentamet® in reducing the impact of X. f. subsp. pauca to the local olive endotherapy performed at the base of the tree using disposable syringes and different cultivarsdoses of theOgliarola biocomplex salentina (Figure and6 )[Cellina33]. di Nardò. Subsequent trials performed at Can- nole andIn the Galatone mid-term duri evaluation,ng 2019 represented the tree ages a mid-term ranged from evaluation 22 to more of the than pathogen 70 years. con- The troltraditional effectiveness, cultivars having Ogliarola started salentina the regular and Cellina application di Nard ofò wereDentamet® cultivated in those in all farms.olive grovesLeccino during trees werethe previous also present four inand the three Galatone years, farm. respectively. Dentamet ® efficacy was evaluated either through the recording of new wilted twigs in the spring (March), summer (July), and autumn (October), or through quantitative real-time PCR performed in the same seasons on 41 trees by following the procedures established by EPPO. PathogensPathogens 20212021,, 1010,, x 668 FOR PEER REVIEW 8 of 22 8 of 22

FigureFigure 5. 5.Confocal Confocal microscope microscope images images of transverseof transverse sections sections of leaf of (leafA), petiole(A), petiole (B), peduncle (B), peduncle of of the thefruit fruit (C (),C two-year-old), two-year-old twig twig ( (DD),), five-year-old five-year-old twig twig ( E(E),), and and longitudinal longitudinal tangential tangential section section of of five-year-oldfive-year-old twig twig (F) excised(F) excised from from a healthy a healthy olive cv. olive Ogliarola cv. Ogliarola salentina treesalentina sprayed tree with sprayed a safranin- with a saf- O/Dentametranin-O/Dentamet®® mixture. Highmixture. magnification High magnif of theication ray parenchyma of the ray cellsparenchyma (G). Upper cells epidermis (G). Upper (uep), epider- peltatemis (uep), trichome peltate (pt), trichome palisade parenchyma (pt), palisade (pp), parenchyma vascular bundle (pp), (vb) vascular with xylem bundle (xy) (vb) and with phloem xylem (xy) (ph),and lowerphloem epidermis (ph), lower (lep), epidermis epidermis (ep),(lep), parenchyma epidermis (ep), cells (pc),parenchyma central parenchyma cells (pc), central cells (cpc), parenchyma secondarycells (cpc), xylem secondary (sxy), secondary xylem (sxy), phloem secondary (sph), periderm phloem (pe), (sph), broken periderm epidermis (pe), (bep), broken xylem epidermis vessel (bep), (xyv),xylem paratracheal vessel (xyv), parenchyma paratracheal cells (ppc),parenchyma fibers (f), cells ray parenchyma(ppc), fibers cells (f), (rpc),ray parenchyma nucleus (nu). cells Scale (rpc), nu- barscleus = 100(nu).µm Scale (A–F bars) and = 5 100µm (μGm). ( ReproducedA–F) and 5 fromμm ( [G33).]. Reproduced from [33].

AA trend significant that indicates reduction a reduction of both of the field field symptoms during (i.e., thetwig year wilting) in both farmsand pathogen andconcentration for all cultivars within was the observed. leaves were The number observed of wiltedat Veglie. twigs It wereworth higher noting in that March during the and decreased in July and October. Ogliarola salentina and Cellina di Nardò cultivars study, apart from X. f. subsp. pauca infection, the trees faced some adverse climatic events were confirmed to be more sensitive to X. f. subsp. pauca than Leccino. At the end of the season,such as just frost before in January the harvest 2017 time, and only a severe a few newheat wiltedwave twigsduring per summer tree were 2017 recorded [33,35]. It was forobserved all cultivars that at in least both 50 farms to 60% (i.e., of a the maximum canopy of should one to be two present twigs in for the the tree Ogliarola for satisfactory salentinaentry of trees). Dentamet® into the xylem tissue. No zinc and copper residues were found withinQuantitative the oil obtained real-time from PCR trees analyses that demonstratedreceived the biofertilizer a decrease in over the bacterial three years. con- centrationFinally, for allextensive cultivars twig and bothresprouting farms in Julyin severely and in October attacked following trees was Dentamet observed® upon treatmentsendotherapy that performed started at the at beginningthe base of April.the tree The using mean disposable bacterial concentration syringes and in different 4 −1 3 -1 Marchdoses wasof the 1.2 biocomplex× 10 CFU g(Figure, which 6) [33]. decreased to 2.1/2.2 × 10 CFU g in July and October, respectively). Comparison of the three cultivars located at Galatone indicated In the mid-term evaluation, the tree ages ranged from 22 to more than 70 years. The that Leccino trees had a lower bacterial concentration (mean of 9.0 × 102 CFU g−1), in comparisontraditional tocultivars Cellina diOgliarola Nardò (1.7 salentina× 104 CFU and gCellina−1) and di Ogliarola Nardò were salentina cultivated (8.7 × 10in3 all farms. CFULeccino g−1) (Figuretrees were7). The also comparatively present in the healthy Gala statustone offarm. the DentametDentamet®®-treated efficacy trees was was evaluated confirmedeither through by their the yield, recording with a productionof new wilted estimated twigs at in about the spring 18 to 23 (March), Kg of olives summer per (July), and autumn (October), or through quantitative real-time PCR performed in the same seasons on 41 trees by following the procedures established by EPPO. A trend that indicates a reduction of the field symptoms during the year in both farms and for all cultivars was observed. The number of wilted twigs were higher in March and decreased in July and October. Ogliarola salentina and Cellina di Nardò cul- tivars were confirmed to be more sensitive to X. f. subsp. pauca than Leccino. At the end

Pathogens 2021, 10, 668 9 of 22 Pathogens 2021, 10, x FOR PEER REVIEW 9 of 22

tree recorded in autumn 2019 in the farms of Galatone and Cannole, whereas the untreated trees were completely wilted [34]. These results further supported the conclusions from theof firstthe studyseason, and just clearly before showed the that harvestX. f. subsp. time,pauca onlycan a befew controlled new wilted in the twigs olive per tree were grovesrecorded not completely for all cultivars damaged byin the both bacterium farms and (i.e., that a a coexistencemaximum with of theone pathogen to two twigs for the isOgliarola possible. salentina trees).

FigureFigure 6. 6.Recovery Recovery of a singleof a single Ogliarola Ogliarola salentina salentina olive tree olive severely tree damaged severely (more damaged than 80% (more of than 80% of thethe canopy canopy dead) dead) by Xylella by Xylella fastidiosa fastidiosasubsp. paucasubsp.grown pauca in Galatonegrown in (Lecce Galatone province) (Lecce after province) trunk after trunk injectioninjection with with Dentamet Dentamet®® from Aprilfrom 2017April to 2017 July 2017. to July (A) 2017. The tree (A before) The treatment.tree before (B treatment.) Initial (B) Initial resproutingresprouting in May in May of shoots of shoots from the from main the tree main branches tree as branches observed 20 as d observed after treatment. 20 d ( Cafter) New treatment. (C) shootsNew continuedshoots continued to grow during to grow June. during (D) At theJune. end (D of) July,At the part end of the of canopyJuly, part was of developed. the canopy was devel- (Eoped.) Despite (E) the Despite drought the occurring drought during occurring summer during 2017, in September,summer 2017, the tree in showed September, a number the of tree showed a suckersnumber as wellof suckers as part of as the well canopy. as part Reproduced of the canopy. from [33]. Reproduced from [33].

Quantitative real-time PCR analyses demonstrated a decrease in the bacterial con- centration for all cultivars and both farms in July and in October following Dentamet® treatments that started at the beginning of April. The mean bacterial concentration in March was 1.2 × 104 CFU g−1, which decreased to 2.1/2.2 × 103 CFU g-1 in July and October, respectively). Comparison of the three cultivars located at Galatone indicated that Lec- cino trees had a lower bacterial concentration (mean of 9.0 × 102 CFU g−1), in comparison to Cellina di Nardò (1.7 × 104 CFU g−1) and Ogliarola salentina (8.7 × 103 CFU g−1) (Figure 7). The comparatively healthy status of the Dentamet®-treated trees was confirmed by their yield, with a production estimated at about 18 to 23 Kg of olives per tree recorded in autumn 2019 in the farms of Galatone and Cannole, whereas the untreated trees were completely wilted [34]. These results further supported the conclusions from the first study and clearly showed that X. f. subsp. pauca can be controlled in the olive groves not completely damaged by the bacterium and that a coexistence with the pathogen is pos- sible.

Pathogens 2021,, 10,, x 668 FOR PEER REVIEW 1010 of of 22 22

Figure 7. Xylella fastidiosa Figuresubsp. 7.pauca XylellaDNA fastidiosa concentration, subsp. pauca expressed DNA concentration, in CFU equivalents expressed g−1 in of CFU leaf, equivalents determined g for−1 untreated plant (assessed asof timeleaf, 0determined control) and for Dentamet untreated®-treated plant (assessed cultivars Cellinaas time di0 Nardcontrol)ò and and Ogliarola Dentamet®-treated salentina, at Cannole and Galatone (assessedcultivars in Cellina 2019). §di Untreated Nardò and control Ogliarola plants salentin deada, in at 2019, Cannole so the and absence Galatone of concentration(assessed in 2019). data is indicative of plant death.§ Untreated (A,B) Graphical control representation plants dead in of 2019, the bacterial so the ab concentrationsence of concentration over time data of Cellina is indicative di Nard ofò plant death. (A,B) Graphical representation of the bacterial concentration over time of Cellina and Ogliarola salentina at Cannole. (C–E) Graphical representation of the bacterial concentration over time of Leccino, di Nardò and Ogliarola salentina at Cannole. (C–E) Graphical representation of the bacterial Cellina di Nardò and Ogliarola salentina at Galatone. Bacterial concentration is expressed in CFU equivalents g−1 of leaf concentration over time of Leccino, Cellina di Nardò and Ogliarola salentina at Galatone. (**** p ≤ 0.0001 vs. controls).Bacterial Reproduced concentration from [34 is]. expressed in CFU equivalents g−1of leaf (**** p ≤ 0.0001 vs. controls). Reproduced from [34]. It is worth noting that the farms where the mid-term evaluation was carried out are locatedIt is inworth areas noting severely that attacked the farms by where the bacterium the mid-term and areevaluation surrounded was carried by completely out are locatedwithered inolive areas groves. severely attacked by the bacterium and are surrounded by completely withered7. The Reprogramming olive groves. of Metabolic Pathways in the Treated Trees

7. TheAmong Reprogramming the analytical of Metabolic techniques Pathways available forin the detectionTreated Trees of molecules involved in the plant-microbe interaction, metabolomics coupled with mass spectrometry (MS) and nuclearAmong magnetic the analytical resonance techniques (NMR) analysesavailable could for the offer detection several of advantagesmolecules involved for obtain- in theing plant-microbe an overview ofinteraction, all small metabolites,metabolomics and coupled for obtaining with mass robust, spectrometry high-reproducible, (MS) and nuclearspectral magnetic data [36]. resonance The combination (NMR) analyses of NMR could and chemometricsoffer several advantages can be useful for obtaining to charac- anterize overview metabolic of all patterns small metabolites, in plants under and stressfor obtaining conditions, robust, such high as pathogen-reproducible, infections, spec- traland data to understand [36]. The combination plant physiology, of NMR which and isch pivotalemometrics for future can be applications useful to characterize in disease metaboliccontrol [37 patterns,38]. Non-targeted in plants under1H-NMR stress fingerprinting, conditions, such in combination as pathogen with infections, unsupervised and to understand(PCA) and supervisedplant physiology, pattern which recognition is pivotal techniques for future (in applications particular, orthogonalin disease control partial [37,38].least squares-discriminant Non-targeted 1H-NMR analysis), fingerprinting, was used in for combination the first time with by unsupervised Girelli et al. [(PCA)39] in andorder supervised to analyze pattern the response recognition of naturally techniques infected (in olive particular, trees cultivars orthogonal Ogliarola partial salentina least squares-discriminantand Cellina di Nardò analysis),, to the Dentamet was used® fortreatments. the first time In particular, by Girelli theet al. work [39] focusedin order onto analyzethe preliminary the response short-time of naturally effect oninfected the metabolic olive trees profiles cultivars of theOgliarola susceptible salentina cultivars, and Cellinawhich showeddi Nardò, a different to the Dentamet® incidence andtreatments. severity In of particular, disease before the work the treatments. focused on Inter- the

Pathogens 2021, 10, x FOR PEER REVIEW 11 of 22

preliminary short-time effect on the metabolic profiles of the susceptible cultivars, which showed a different incidence and severity of disease before the treatments. Interestingly, unsupervised principal component analysis (PCA) showed, only in the case of Dentam- et®-treated samples, a clear partition of data, with a good grouping according to the original cultivar (Ogliarola salentina and Cellina di Nardò) observed (Figure 8a). On the other hand, the untreated samples submitted to PCA analysis showed no differences in the metabolomic profiles of the two cultivars (Figure 8b). These results seemed to suggest that the effect of the presence of a pathogen, such as X. f. subsp. pauca on the metabolic profile of naturally infected plants samples, could be predominant with respect to the differences normally observed among the olive cultivars. The effect of the biocomplex treatment resulted into specific involvement of xylematic polyphenols and carbohydrates for each of the two studied cultivars. In particular, treated Cellina di Nardò trees showed a higher polyphenols content, whereas treated Ogliarola salentina trees showed a higher Pathogens 2021, 10, 668sugar content. 11 of 22 The metabolic response of both infected and treated adult olive cultivars Ogliarola salentina and Cellina di Nardò was then investigated in two sampling periods performed during the first year of the treatments with Dentamet® to the olive crown [40]. The estingly, unsupervised principal component analysis (PCA) showed, only in the case of 1H-NMR metabolomic approach, in conjunction with a multivariate statistical analysis, Dentamet®-treated samples, a clear partition of data, with a good grouping according to showed that for both these X. f. subsp. pauca-susceptible cultivars, some metabolites such the original cultivar (Ogliarola salentina and Cellina di Nardò) observed (Figure8a). On as quinic acid, the aldehydic form of oleuropein, ligstroside and phenolic compounds, the other hand, the untreated samples submitted to PCA analysis showed no differences in were consistentlythe metabolomicfound as discrimina profilestive of the for two the cultivars untreated (Figure trees8 b).( Figure Theses results 9 andseemed 10). to suggest Quinic acid, athat precursor the effect of lignin, of the presencewas confirmed of a pathogen, as a disease such biomarker as X. f. subsp. for thepauca oliveon the metabolic trees infected byprofile X. f. ofsubsp. naturally pauca infected, as previously plants observed samples, for could the besame predominant cultivars [41,42] with respect to the and for X. f. subsp.differences fastidiosa normally-infected observed grapevines among [43]. The the olivetwo cultivars cultivars. showed The effect a distinct of the biocomplex response to thetreatment biocomplex resulted treatment. into specific A consistent involvement increase of xylematicin malic acid polyphenols was observed and carbohydrates for the Ogliarolafor eachsalentina of the trees, two studiedwhereas cultivars. in the Cellina In particular, di Nardò treated trees, Cellina the treatments di Nardò trees showed seemed to inducea higher the accumulation polyphenols content,of γ−aminobutyrate whereas treated (GABA), Ogliarola as already salentina described trees showedas a higher a response to environmentalsugar content. strain and considered as an adaptive stress-mitigating factor [44,45].

A) Untreated samples B) Treated samples

Figure 8. 1H-NMR fingerprinting in combination with unsupervised principal component analysis score plots for Ogliarola Figure 8. 1H-NMR fingerprinting in combination with unsupervised principal component analysis ® ® salentina andscore Cellina plots di for Nard Ogliarolaò olive salentina cultivars. and (A) DentametCellina di Nardò-untreated olive samples;cultivars. ( B(A) Dentamet) Dentamet®-untreated-treated samples. Repro- duced fromsamples; [39]. (B) Dentamet®-treated samples. Reproduced from [39].

Recently, a 1H-NMRThe metabolic metabolomic response approach of both was infected used andto analyze treated the adult xylematic olive cultivars ex- Ogliarola salentina and Cellina di Nardò was then investigated in two sampling periods performed tracts of the X. f. subsp. pauca-tolerant cultivar Leccino in comparison® with the susceptible 1 cultivars Ogliaroladuring salentina the first and year Cellina of the treatments di Nardò withfollowing Dentamet a mid-termto the olive period crown of Den- [40]. The H-NMR metabolomic approach, in conjunction with a multivariate statistical analysis, showed that tamet® spray treatment (i.e., three years of spray treatments during spring and early for both these X. f. subsp. pauca-susceptible cultivars, some metabolites such as quinic acid, autumn). The metabolic profiles evaluation focused in a specific time span (six months the aldehydic form of oleuropein, ligstroside and phenolic compounds, were consistently found as discriminative for the untreated trees (Figures9 and 10). Quinic acid, a precursor of lignin, was confirmed as a disease biomarker for the olive trees infected by X. f. subsp. pauca, as previously observed for the same cultivars [41,42] and for X. f. subsp. fastidiosa- infected grapevines [43]. The two cultivars showed a distinct response to the biocomplex treatment. A consistent increase in malic acid was observed for the Ogliarola salentina trees, whereas in the Cellina di Nardò trees, the treatments seemed to induce the accumulation of γ–aminobutyrate (GABA), as already described as a response to environmental strain and considered as an adaptive stress-mitigating factor [44,45]. Pathogens 2021, 10, x FOR PEER REVIEW 12 of 22

treatment April to September after six months suspension), with the aim to evaluate any possible memory effects due to previous treatments persisting after suspension periods. Therefore, metabolomic analyses were performed on leaf sample extracts from infected and untreated trees in two specific samplings (March, before the start and October after the end of treatments) over the last treatment year [46]. Statistical models, obtained for untreated and treated samples, respectively, demonstrated a different degree of separation, more pronounced in the second sampling, essentially related to the higher mannitol content in the treated samples. The intracellular accumulation of mannitol can be considered as a strategy to improve tolerance against water deficit thanks to its osmoprotectant and antioxidant ability to protect chloroplast [47,48]. Moreover, as already reported [49], the accumulation of mannitol was observed in olive leaves in response to foliar fertilization, suggesting an improvement of physio- logical performance and the photosynthetic capability of olive trees. For the untreated samples, a higher relative content of phenolic compounds such as tyrosol and hydroxy- tyrosol moieties of oleuropein and its aldehydic forms and quinic acid were observed in the second sampling for all the analyzed cultivars, consistent with previous findings

Pathogens 2021, 10,[39,50]. 668 The analyses of the specific influence of the sampling period on metabolic profiles 12 of 22 for infected cultivars indicated a specific rank (i.e., Leccino > Cellina di Nardò > Ogliarola salentina) for the observed discrimination between the two sampling periods [46].

(A) (B)

Figure 9. (A) Orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot for Ogliarola salentina treated Figure 9. (A) Orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot for ® with DentametOgliarola(green salentina triangles) treated and with untreated Dentamet® (control) (green (red triangles) triangles) and leaf untreated extracts (control) samples (red (1 + triangles) 3 + 0; R2X = 0.613; R2Y = 0.815leaf; Q2 extracts = 0.418); samples (B) S-line (1 plot+ 3 + for 0; theR2X model, = 0.613; indicating R2Y = 0.815; molecular Q2 = 0.418); components (B) S-line responsible plot for the for model, the class separation. PathogensThe 2021 corresponding, 10, x FOR PEER predictive REVIEW loadings are colored according to the correlation scaled loading [p(corr)]. Reproduced13 of 22 indicating molecular components responsible for the class separation. The corresponding predic- from [40]. tive loadings are colored according to the correlation scaled loading [p(corr)]. Reproduced from [40].

(A) (B)

Figure 10. (A) Orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot for Cellina di Nardò treated ® Figure 10. (A) Orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot for with Dentamet (green circles)Cellina and untreateddi Nardò (control)treated with (red circles)Dentamet® leaf extracts(green samplescircles) and (1 + untreated 1 + 0; R2X (control) = 0.567; R2Y (red = circles) 0.957; Q2 = 0.897); (B) S-lineplot forleaf theextracts model, samples indicating (1 + 1 molecular + 0; R2X components= 0.567; R2Y responsible= 0.957; Q2 for= 0.897); the class (B) S-lineplot separation. for The the corresponding predictive loadingsmodel, areindicating colored accordingmolecular to components the correlation resp scaledonsible loading for the [p(corr)]. class separation. Reproduced The from corre- [40]. sponding predictive loadings are colored according to the correlation scaled loading [p(corr)]. 1 ReproducedRecently, from a [40].H-NMR metabolomic approach was used to analyze the xylematic extracts of the X. f. subsp. pauca-tolerant cultivar Leccino in comparison with the susceptible culti- vars OgliarolaMoreover, salentina an increasing and Cellina intraclass di Nard discriminationò following abetween mid-term treated period and of Dentamet untreated® spraysample treatment groups could (i.e., threebe reported years offor spray all the treatments cultivars in during the second spring sampling, and early suggesting autumn). the occurrence of different levels of disease, season and treatment effects over the con- sidered time span. A significant differentiation among treated and untreated samples also in the first sampling was observed only for Leccino, suggesting a possible memory effect of the previous year treatment. The comparison of tolerant Leccino with respect to the susceptible cultivars Ogliarola salentina and Cellina di Nardò, grouped as a single class, demonstrated a general smoothing of the observed differences in the second with respect to the first sampling for both treated and untreated samples. Higher amounts of flavonoids were observed both in infected untreated and treated Leccino cultivar. On the contrary, in the susceptible cultivars, variables ascribable to mannitol signals and indicating a higher relative sugar content with respect to Leccino were observed (Figure 11). The observed differences in the infected Leccino with respect to susceptible cultivars showed interesting reversed behavior for mannitol levels with respect to the analogous comparison for healthy trees (Table 1). Indeed, Leccino healthy leaf samples showed a higher content of mannitol and sucrose with respect to the healthy susceptible cultivars—Ogliarola salentina and Cellina di Nardò. The high constitutive sucrose content, observed for Leccino cultivar, could also be involved in the X. f. subsp. pauca resistance due to sucrose’s role as a signaling molecule in response to cavitation [42].

Pathogens 2021, 10, 668 13 of 22

The metabolic profiles evaluation focused in a specific time span (six months treatment April to September after six months suspension), with the aim to evaluate any possible memory effects due to previous treatments persisting after suspension periods. There- fore, metabolomic analyses were performed on leaf sample extracts from infected and untreated trees in two specific samplings (March, before the start and October after the end of treatments) over the last treatment year [46]. Statistical models, obtained for untreated and treated samples, respectively, demon- strated a different degree of separation, more pronounced in the second sampling, es- sentially related to the higher mannitol content in the treated samples. The intracellular accumulation of mannitol can be considered as a strategy to improve tolerance against wa- ter deficit thanks to its osmoprotectant and antioxidant ability to protect chloroplast [47,48]. Moreover, as already reported [49], the accumulation of mannitol was observed in olive leaves in response to foliar fertilization, suggesting an improvement of physiological perfor- mance and the photosynthetic capability of olive trees. For the untreated samples, a higher relative content of phenolic compounds such as tyrosol and hydroxytyrosol moieties of oleuropein and its aldehydic forms and quinic acid were observed in the second sampling for all the analyzed cultivars, consistent with previous findings [39,50]. The analyses of the specific influence of the sampling period on metabolic profiles for infected cultivars indicated a specific rank (i.e., Leccino > Cellina di Nardò > Ogliarola salentina) for the observed discrimination between the two sampling periods [46]. Moreover, an increasing intraclass discrimination between treated and untreated sample groups could be reported for all the cultivars in the second sampling, suggesting the occurrence of different levels of disease, season and treatment effects over the considered time span. A significant differentiation among treated and untreated samples also in the first sampling was observed only for Leccino, suggesting a possible memory effect of the previous year treatment. The comparison of tolerant Leccino with respect to the susceptible cultivars Ogliarola salentina and Cellina di Nardò, grouped as a single class, demonstrated a general smoothing of the observed differences in the second with respect to the first sampling for both treated and untreated samples. Higher amounts of flavonoids were observed both in infected untreated and treated Leccino cultivar. On the contrary, in the susceptible cultivars, variables ascribable to mannitol signals and indicating a higher relative sugar content with respect to Leccino were observed (Figure 11). The observed differences in the infected Leccino with respect to susceptible cultivars showed interesting reversed behavior for mannitol levels with respect to the analogous comparison for healthy trees (Table1). Indeed, Leccino healthy leaf samples showed a higher content of mannitol and sucrose with respect to the healthy susceptible cultivars—Ogliarola salentina and Cellina di Nardò. The high constitutive sucrose content, observed for Leccino cultivar, could also be involved in the X. f. subsp. pauca resistance due to sucrose’s role as a signaling molecule in response to cavitation [42]. The overall results highlighted how the infection caused by X. f. subsp. pauca strongly modifies the metabolism of olive trees, and how a zinc-copper-citric acid biocomplex can induce an early reprogramming of the metabolic pathways in the infected trees. The different responses to Dentamet® treatment would seem to be correlated to olive cultivars physiology and/or the pathogen attack levels. Pathogens 2021, 10, 668 14 of 22 Pathogens 2021, 10, x FOR PEER REVIEW 14 of 22

Figure 11. Magnitude scale of the levels of discriminating metabolites comparison between untreated and treated olive Figure 11. Magnitude scale of the levels of discriminating metabolites comparison between untreated and treated olive treestrees inin twotwo samplingsampling periodsperiods (March(March andand October).October). OgliarolaOgliarola salentinasalentina (((AA,D) ),and Cellina (D)), diCellina Nard òdi( BNardò,E) and, ((B Leccino) and (E (())C and,,F)) cultivarsLeccino (( providedC) and (F)) as cultivars values of provided−Log2 (FC). as values Metabolites of −Log2 with (FC).− MetabolitesLog2 (FC) negative with −Log2 values (FC) have negative higher values concentration have higher in untreatedconcentration samples, in untreated while − samples,Log2 (FC) while positive −Log2 values (FC) indicated positive values metabolites indicated with metabolites higher concentration with higher treated concentration samples. Statisticaltreated samples. significance Statistical (one-way significance analysis (one-way of variance analys (ANOVA))is of variance was set (ANOVA)) at least at adjustedwas set atp-values least at < adjusted 0.05 and p indicated-values < with0.05 and 0 ‘***’ indicated 0.001 ‘**’0.01 with ‘*’0 ‘***’00.05 0.0010. Reproduced ‘**’0.01 ‘*’ ′ from0.05 ′. [ 46Reproduced]. from [46].

1 TableTable 1.1. MainMain metabolitesmetabolites identifiedidentified through 1H-NMRH-NMR metabolomic metabolomic analyses analyses as as discriminating discriminating in in leaf leaf extracts extracts samples samples of ofolive olive cultivars cultivars Ogliarola Ogliarola salentina, salentina, Cellina Cellina di di Nardò Nardò andand Leccino Leccino naturally naturally infected infected by X. f. subsp.subsp. pauca, treatedtreated withwith Dentamet® or healthy. Based on [39,40.46]. Note quinic acid as a biomarker for naturally infected trees, and mannitol for Dentamet® or healthy. Based on [39,40,46]. Note quinic acid as a biomarker for naturally infected trees, and mannitol for treated and healthy Leccino trees. treated and healthy Leccino trees. Infected Healthy Ogliarola InfectedCellina Leccino Ogliarola Healthy Cellina Leccino UntreatedOgliarola Treated Untreated Cellina Treated Untreated Leccino Treated Ogliarola Untreated CellinaUntreated LeccinoUntreated Oleuropein Untreated Treated Untreated Treated Untreated Treated Untreated Untreated Untreated derivatives Oleuropein   derivativesGlucose XXXXXX Sucrose  Glucose Flavonoids XX  SucroseMannitol    X FlavonoidsMalate  X Quinic acid    Mannitol XXXX Malate X The overall results highlighted how the infection caused by X. f. subsp. pauca Quinic strongly modifies the metabolism of olive trees, and how a zinc-copper-citric acid bio- acid XXXcomplex can induce an early reprogramming of the metabolic pathways in the infected trees. The different responses to Dentamet® treatment would seem to be correlated to olive cultivars physiology and/or the pathogen attack levels. 8. The Control Strategy at Work 8. The Control Strategy at Work In addition to the spraying of Dentamet® onto the olive tree crown once per month fromIn spring addition to autumn, to the spraying the X. f.of Dentamet®subsp. pauca ontocontrol the olive strategy tree crown employs once agronomical per month measuresfrom spring to reduce to autumn, the population the X. f. of subsp. the insect pauca vector control (Philaenus strategy spumarius employs) in surroundingagronomical measures to reduce the population of the insect vector (Philaenus spumarius) in sur-

Pathogens 2021, 10, 668 15 of 22

vegetation and to maintain a regular shape of the olive tree crown by regular pruning. Maintaining or restoring good soil fertility is also important for the efficacy of the strategy. The vector density can reach very high levels in the spring, when 10 to 40 individual nymphs of P. spumarius can be found on each m2 of soil hosting wild plant species [51,52]. From the end of spring to early autumn, P. spumarius adults can reach the young olive leaves and release X. f. subsp. pauca directly into the leaf xylem. Mechanical removal of weeds by harrowing during this period reduces the risk of pathogen spread within and between the olive groves. This is enforced by the Apulia region officers, who impose a fine for failure to remove weeds at the end of winter into spring. Regular pruning of the olive tree every one to two years maintains an equilibrium between vegetative growth and fruit production, and facilitates pest control and har- vest [53,54]. The distribution of a foliar spray such as of Dentamet® is facilitated by a well-aerated canopy, and thorough coverage of the leaves augments the efficacy of the treatment. Hard pruning, performed on a four- to five-year basis, was the norm during the last decades in most of the Salento olive groves, but this resulted in shading, a hap- hazard spatial distribution of the canopy, shorter and more compact vegetative growth, and deadwood in the inner part of the tree, ultimately leading to decreased tree longevity and lower productivity [55,56]. Contrary to an effective reduction of X. f. subsp. pauca density within citrus trees obtained in Brazil with pruning [57], the attempts to reduce the bacterium inoculum within olive trees of Salento through severe pruning caused the death of the tree during the following months [33,56]. Thus, light pruning aiming at maintaining just a physiological equilibrium of the tree between the vegetative and productive phases should be now performed in Salento. Maintenance of good fertility and beneficial microbial communities in the soil is important in pathogen control [58]. Depletion of zinc, copper, and manganese content has been observed in the soil of Salento, where the OQDS symptoms are very severe. Deficiency in these ions was also observed in the leaves of diseased olive trees that grow in this area [59,60]. In areas north of the infected zone (i.e., north of the Bari areas), a higher zinc and copper content both in soil and olive leaves is found in spite of the lithological matrix (calcareous soils) being similar in the two areas [59]. Depletion of zinc and copper in the Salento soil may explain the effectiveness of Dentamet® in restoring good vegetative growth, since zinc and copper are involved in many plant metabolic pathways other than plant defense mechanisms [61,62]. Apart from micronutrients formulations, beneficial microorganisms are also important for augmenting resistance to X. f. subsp. pauca [33].

9. A Novel Endotherapy to Fine-Tune the Tree Recovery Bacterial pathogens on the leaf surface of plants can be effectively controlled by direct contact with copper applied by foliar sprays [63,64]. With xylem-invading pathogens such as Xylella fastidiosa, the antimicrobial compounds must contact the pathogen within the xylem vessels. This is more likely to occur through injection than through foliar ap- plication or soil drenching [65]. Delivery of antimicrobials by injection into the plant’s vascular system (endotherapy) offers multiple advantages compared to foliar applica- tions, especially in diseases such as olive quick decline syndrome that are caused by xylem-restricted pathogens. Endotherapy enhances the effectiveness of the treatment by delivering the compound directly to the pathogen, and accelerates translocation of the injected antimicrobial compounds, even in heavily infected plants with limited re- maining foliage. Endotherapy avoids the unintended dissipation of the antimicrobial compound into the air and the broader ecosystem (closed application system) and therefore significantly improves worker and bystander safety by minimizing dermal and inhala- tory exposure (https://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/ occupational-pesticide-handler-exposure-data#phed). Finally, endotherapy reduces the amount of active ingredients that are applied and reduces the overall environmental load when compared to foliar application methods. Pathogens 2021, 10, x FOR PEER REVIEW 16 of 22

ingredients that are applied and reduces the overall environmental load when compared Pathogens 2021, 10, 668 16 of 22 to foliar application methods. The use of traditional endotherapy methods has been of limited use due to several important constraints. The primary constraint is that many systems are based on drilling sizableThe holes use of in traditional the trunk (4 endotherapy to 12 mm, 5 methods to 120 mm has depth) been of [66,67], limited leading use due to tosignificant several importantplant damage constraints. and potential The primary entry points constraint for issecondary that many infections systems arewith based other on pathogens drilling sizable[68]. Notably, holes in repeated the trunk treatments (4 to 12 mm, of infected 5 to 120 plants mm depth) can lead [66 to,67 a], negative leading toimpact significant on tree planthealth damage due to and less potential effective entry wound points closure for secondary and compartmentalization infections with other [69,70]. pathogens A second [68]. Notably,important repeated constraint treatments is that of the infected formulated plants active can lead ingredient to a negative is delivered impact on to tree the health heart- duewood to lessof the effective trunk, wound which closurepresumably and compartmentalization leads to a slower distribution [69,70]. A of second the active important ingre- constraintdient(s) throughout is that the formulated the plant. activeThe heartwood ingredient has is delivered no or at to least the heartwooda limited physiological of the trunk, whichrole, in presumably contrast to leadsthe active to a slower xylem, distribution which transports of the activewater ingredient(s)and nutrients throughout throughout the the plant.plant The[71]. heartwood Additionally, has noit was or at observed least a limited that the physiological sap flux decreases role, in contrastsignificantly to the towards active xylem,the heartwood which transports boundary water [72]. and In nutrientsaddition, throughoutthe traditional the plantsystems [71 ].have Additionally, operational it was lim- observeditations limiting that the sapthe fluxnumber decreases of trees significantly that can be towards treated theby heartwoodone person boundaryto fewer than [72]. 100 In addition,per day. the traditional systems have operational limitations limiting the number of trees that canIn July be treated 2020, a by proprietary one person injection to fewer system than 100 produced per day. by Invaio Sciences was used to injectIn 30 July trees 2020, in athe proprietary Salento area injection with a system micronutrient produced containing by Invaio Scienceszinc and wascopper used ions. to injectThe Invaio 30 trees system in the includes Salento areaan extremely with a micronutrient thin, arrowhead-shaped containing zincinjection and coppertip, resulting ions. Thein minimal Invaio system invasiveness includes and an extremelyplant damage thin, arrowhead-shaped(Figures 12 and 13). injection The tip tip, is resultingonly 12 mm in minimallong and invasiveness 1.5 mm thick, and with plant a size damage and (Figuresshape designed 12 and 13for). the The precise tip is only and 12effective mm long de- andlivery 1.5 of mm treatment thick, with formulations a size and shapeto the designedtree’s active for vasculature. the precise and The effective formulations delivery are ofinjected treatment at low formulations pressure (between to the tree’s 1 and active 3 bars vasculature.) directly Theinto formulationsthe vascular aresystem, injected thus atenhancing low pressure uptake (between and distribu 1 and 3tion bars) dynamics directly intoof the the injected vascular compound. system, thus The enhancing injection uptakesystem and anddistribution its operating dynamics parameters of theenable injected a higher compound. throughput The of injection injections system compared and itsto operatingtraditional parameters systems. Furthermore, enable a higher the throughput tip is designed of injections to avoid compared clogging, toallowing traditional effi- systems.cient delivery. Furthermore, We are thealso tip investigating is designed towhether avoid clogging,multiple injections allowing efficient with the delivery. same tip We are arepossible. also investigating whether multiple injections with the same tip are possible.

TM FigureFigure 12. 12.Prototype Prototype of of Invaio Invaio Sciences Sciences TIPS TIPSTM injectioninjection system system applied applied to to an an olive olive tree. tree. The The system system allowsallows a a low-pressure low-pressure release release of of compounds. compounds.

Pathogens 2021, 10, 668 17 of 22 Pathogens 2021, 10, x FOR PEER REVIEW 17 of 22

FigureFigure 13. 13.Comparison Comparison traditional traditional injection injection system system and and Invaio’s Invaio’s injection injection system system (based (based on on internal internal Invaio Invaio Sciences Sciences data data and insights). and insights). 10. Considerations and Perspectives 10. Considerations and Perspectives A great part of the Salento territory has been facing a dramatic decline of olive health A great part of the Salento territory has been facing a dramatic decline of olive health during this last decade [25]. Records of OQDS symptoms document the spread of X. f. during this last decade [25]. Records of OQDS symptoms document the spread of X. f. subsp. pauca from its initial invasion in the Gallipoli area about from 2008 [22], so that subsp. pauca from its initial invasion in the Gallipoli area about from 2008 [22], so that within barely 15 years, a multi-millennial agro-ecosystem is at risk of extinction, with a within barely 15 years, a multi-millennial agro-ecosystem is at risk of extinction, with a subsequent impoverishment of a historical, social, economic, and landscape heritage [26]. subsequent impoverishment of a historical, social, economic, and landscape heritage [26]. In recent years, we have shown that a coexistence with X. f. subsp. pauca is possible In recent years, we have shown that a coexistence with X. f. subsp. pauca is possible in in the so-called “infected area” of Salento (i.e., the province of Lecce, and part of the the so-called “infected area” of Salento (i.e., the province of Lecce, and part of the provinces provinces of Brindisi and Taranto). The tree crown nebulization of Dentamet® (a bio- of Brindisi and Taranto). The tree crown nebulization of Dentamet® (a biocomplex contain- complex containing zinc, copper, and citric acid) during spring, summer, and early au- ing zinc, copper, and citric acid) during spring, summer, and early autumn significantly tumn significantly reduced both the symptoms (i.e., twig and branch wilting) and the reduced both the symptoms (i.e., twig and branch wilting) and the bacterial cell density bacterial cell density within the leaves of the sensitive olive cultivars Ogliarola salentina within the leaves of the sensitive olive cultivars Ogliarola salentina and Cellina di Nardò as measuredand Cellina by quantitativedi Nardò as real-time measured PCR by [ 33quantitative,34]. Dentamet real-time® treatments PCR [33,34]. resulted Dentamet® in a rapid reprogrammingtreatments resulted of some in a rapid indicative reprogramming metabolic pathways, of some indicative as observed metabolic by metabolomics, pathways, as whichobserved re-established by metabolomics, a more normal which treere-established physiology [a39 ,more40,46 ].normal The yearly tree amountphysiology of copper[39,40,46]. released The inyearly the environment amount of copper by means rele ofased this in control the environment strategy satisfies by means the current of this requirementscontrol strategy aimed satisfies at avoiding the current the accumulationrequirements aimed of copper at avoiding in the soil the [73 accumulation]. It has been of calculatedcopper in thatthe soil the [73]. amount It has of been copper calculated and zinc that released the amount in the of soil copper in one and year zinc by released the six treatmentsin the soil isin 0.06one ppmyear andby the 0.12 six ppm, treatmen respectively.ts is 0.06 Finally,ppm and an 0.12 innovative ppm, respectively. endotherapy Fi- approachnally, an appearsinnovative to provide endotherapy a more approach efficient techniqueappears to for provide promoting a more regrowth efficient in technique severely damagedfor promoting trees andregrowth reducing in severely the amount damaged of product trees and required reducing to the treat amount a tree of and product thus reducingrequired the to treat cost ofa tree the controland thus strategy reducing as well.the cost of the control strategy as well. Collectively,Collectively, these these results results demonstrate demonstrate an an efficient, efficient, simple, simple, low-cost, low-cost, andand environ-environ- mentallymentally sustainable sustainable strategy strategy to to control controlX. X. f. f.subsp. subsp.pauca paucain in Salento, Salento, at at least least in in the the areas areas notnot already already completely completely destroyed destroyed by by the the bacterium. bacterium. The The complete complete restoration restoration of of the the olive olive agroecosystemagroecosystem is is no no longer longer possible, possible, but but the the application application of of the the measures measures herein herein described described onon a a large large scale scale could could allow allow some some preservation preservation of it,of withoutit, without sacrificing sacrificing the typicalthe typical Apulian Apu- olivelian olive growing growing cultivars, cultivars, such assuch Cellina as Cellina di Nard di òNardòand Ogliarola and Ogliarola salentina. salentina. Despite Despite the generalthe general opinion opinion that it that was it not was possible not possible to control to control the bacterium, the bacterium, a number a number of farmers of farmers of the provincesof the provinces of Lecce, of Brindisi, Lecce, Brindisi, and Taranto and are Tarant applyingo are theapplying control the strategy control herein strategy described, herein thisdescribed, representing this representing valid examples valid of examples resilience of for resilience the territory. for the Within territory. this concept,Within this in Salento,concept, Slow in Salento, Food has Slow promoted Food has two promoted clusters two of resilient clusters farmers of resilient to enhance farmers their to enhance work andtheir attest work to theirand attest perseverance to their inperseverance working to preservein working the localto preserve heritage. the Apart local from heritage. this initiative,Apart from currently, this initiative, there are currently, more than there 700 haare ofmore olive than groves 700 in ha the of provincesolive groves of Lecce, in the Brindisi,provinces and of TarantoLecce, Brindisi, where thisand strategyTaranto iswher beinge this applied strategy to controlis beingX. applied f. subsp. to controlpauca (FiguresX. f. subsp. 14 and pauca 15 ).( Figures 14 and 15).

PathogensPathogens 2021,, 102021,, xx, FOR10FOR, 668 PEERPEER REVIEWREVIEW 18 of 2218 of 22

Figure 14. A comparisonFigure between 14. a non-treatedA comparison (on the between left) and a treated non-treated olive grove (on (onthe the left) right) and located treated in theolive Otranto grove area (on the (Lecce province; coordinates:right)right) Lat. locatedlocated 40.138095; inin thethe Lon. OtrantoOtranto 18.467825) areaarea within(Lecce(Lecce the provprov “infected”ince;ince; coordinates:coordinates: area. It is evident Lat.Lat. 40.138095;40.138095; to see the capability Lon.Lon. 18.467825)18.467825) of tree restoration providedwithin by the the application “infected” of thearea. control It is evident strategy. to see the capability of tree restoration provided by the application of the control strategy.

Figure 15. An olive groveFigure located 15. in An Nard oliveò (Lecce grove province; located coordinates:in Nardò (Lecce Lat. 40.187637; province; Lon. coordinates: 17.987413) Lat. that, 40.187637; since 2016, Lon. has been following the control17.987413) strategy that, described since 2016, in the has text. been It is locatedfollowing in the the “infected” control strategy area and bordersdescribed a territory in the withtext. It is dead or very damaged olivelocatedlocated trees. inin thethe “infected”“infected” areaarea andand bordersborders aa teterritoryrritory withwith deaddead oror veryvery damageddamaged oliveolive trees.trees.

AuthorAuthor Contributions Contributions: Conceptualization,Conceptualization, M.S.,M.S., S.L., S.L., D.V., D.V., and and F.P.F.; F.P.F.; resources, resources, M.S., M.S., S.L., D.V. S.L., and D.V. andF.P.F.; F.P.F.; data data curation, curation, M.S., M.S., S.L., S.L., N.P., N.P., V.S., V.S., G.T., G.T., D.V., M.O.,D.V., M.O., U.W., J.M.C.,U.W., J.M.C., P.H., M.D.C., P.H., M.D.C., F.A., D.M., F.A., L.D.C., C.R.G. and F.P.F.; writing—original draft preparation, M.S.; S.L., D.V., C.R.G. and F.P.F.; D.M., L.D.C., C.R.G and F.P.F.; writing—original draft preparation, M.S.; S.L., D.V., C.R.G. and writing—review and editing, M.S., S.L., D.V., G.C., C.R.G., G.D. and F.P.F.; funding acquisition, F.P.F.; writing—review and editing, M.S., S.L., D.V., G.C., C.R.G., G.D. and F.P.F.; funding acquisi- tion,tion, M.S.,M.S., S.L.,S.L., D.V.,D.V., G.C.,G.C., andand F.P.F.F.P.F. AllAll authorsauthors have read and agreed to the published version of thethe manuscript.manuscript. Funding This study was funded by the Apulia region agreement: ””Strategie di controllo integrato per il contenimento di Xylella fastidiosa inin olivetioliveti pugliesipugliesi eded analisianalisi epidemiologicheepidemiologiche deldel complessocomplesso del disseccamento rapido dell’olivo (CoDiRO)”, and by thethe projectsprojects fundedfunded byby thethe ItalianItalian MinistryMinistry of Agriculture, Food and Forestry: “OLIvicoltura e Difesa da Xylella fastidiosa ee dada InsettiInsetti vettorivettori inin

Pathogens 2021, 10, 668 19 of 22

M.S., S.L., D.V., G.C., and F.P.F. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by the Apulia region agreement: “Strategie di controllo integrato per il contenimento di Xylella fastidiosa in oliveti pugliesi ed analisi epidemiologiche del complesso del disseccamento rapido dell’olivo (CoDiRO)”, and by the projects funded by the Italian Ministry of Agriculture, Food and Forestry: “OLIvicoltura e Difesa da Xylella fastidiosa e da Insetti vettori in Italia” (Oli.Di.X.I.It D.M. 23773 del 6/09/2017), “Salvaguardia evalorizzazione del patrimonio olivicolo italiano con azioni di ricerca nel settore della difesa fitosani-taria (SALVAOLIVI)”. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. Conflicts of Interest: The co-authors—D.V., M.O., U.W., J.M.C. and P.H.—are employees of Inavio Sciences, which has pending patent applications related to the injection technologies described in this paper.

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