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

Abstract

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.

Keywords: NMR metabolomic; endotherapy; olive quick decline syndrome; real-time PCR; sustainable development goals of the United Nations.

Figure 1

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

Figure 2

A continuum of olive trees that extend over kilometers characterizes the multi-millennial olive agro-ecosystem of Salento (Apulia, Italy).

Figure 3

Bactericidal evaluation of Xylella fastidiosa subsp. pauca strain De Donno CFBP8402 (A), X. f. subsp. fastidiosa strain Temecula1 (B), and X. f. subsp. multiplex strain CFBP 8416 (C). Aliquots of 10 μL and 10-fold dilutions from 10⁷ to 10³ CFU mL⁻¹ of each X. fastidiosa subspecies were spotted on PD2 medium agar plates supplemented or not with 1:10, 1:50 and 1:100 Dentamet^® dilutions and incubated at 28 °C. Ctr (control): X. fastidiosa cultures grown on PD2 medium Dentamet^®-free. Reproduced from [34].

Figure 4

Biofilm assay 30 dpi of Xylella fastidiosa subsp. pauca (De Donno_CFBP8402) (Xfp) (A), and 15 dpi of X. fastidiosa subsp. fastidiosa (Temecula1) (Xff) (B) and X fastidiosa subsp. multiplex (CFBP 8416) (Xfm) (C). The ordinate axes report % of biofilm formation, the abscissa axes the Dentamet^® dilutions. Values are means ± SD of three independent biological replicates (n = 7). A statistically significant difference was obtained between Xfp, Xff and Xfm PD2 cultures (untreated control) and 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). Reproduced from [34].

Figure 5

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

Figure 6

Recovery of a single Ogliarola salentina olive tree severely damaged (more than 80% of the canopy dead) by Xylella fastidiosa subsp. pauca grown in Galatone (Lecce province) after trunk injection with Dentamet^® from April 2017 to July 2017. (A) The tree before treatment. (B) Initial resprouting in May of shoots from the main tree branches as observed 20 d after treatment. (C) New shoots continued to grow during June. (D) At the end of July, part of the canopy was developed. (E) Despite the drought occurring during summer 2017, in September, the tree showed a number of suckers as well as part of the canopy. Reproduced from [33].

Figure 7

Xylella fastidiosa subsp. pauca DNA concentration, expressed in CFU equivalents g−1 of leaf, determined for untreated plant (assessed as time 0 control) and Dentamet^®-treated cultivars Cellina di Nardò and Ogliarola salentina, at Cannole and Galatone (assessed in 2019). § Untreated control plants dead in 2019, so the absence of concentration data is indicative of plant death. (A,B) Graphical representation of the bacterial concentration over time of Cellina di Nardò and Ogliarola salentina at Cannole. (C–E) Graphical representation of the bacterial concentration over time of Leccino, Cellina di Nardò and Ogliarola salentina at Galatone. Bacterial concentration is expressed in CFU equivalents g−1 of leaf (**** p ≤ 0.0001 vs. controls). Reproduced from [34].

Figure 8

¹H-NMR fingerprinting in combination with unsupervised principal component analysis score plots for Ogliarola salentina and Cellina di Nardò olive cultivars. (A) Dentamet^®-untreated samples; (B) Dentamet^®-treated samples. Reproduced from [39].

Figure 9

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

Figure 10

(A) Orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot for Cellina di Nardò treated with Dentamet^® (green circles) and untreated (control) (red circles) leaf extracts samples (1 + 1 + 0; R2X = 0.567; R2Y = 0.957; Q2 = 0.897); (B) S-lineplot for the model, indicating molecular components responsible for the class separation. The corresponding predictive loadings are colored according to the correlation scaled loading [p(corr)]. Reproduced from [40].

Figure 11

Magnitude scale of the levels of discriminating metabolites comparison between untreated and treated olive trees in two sampling periods (March and October). Ogliarola salentina (A,D), Cellina di Nardò (B,E) and, Leccino ((C,F)) cultivars provided as values of −Log2 (FC). Metabolites with −Log2 (FC) negative values have higher concentration in untreated samples, while −Log2 (FC) positive values indicated metabolites with higher concentration treated samples. Statistical significance (one-way analysis of variance (ANOVA)) was set at least at adjusted p-values < 0.05 and indicated with 0 ‘***’ 0.001 ‘**’0.01 ‘*’ ′0.05 ′. Reproduced from [46].

Figure 12

Prototype of Invaio Sciences TIPS^TM injection system applied to an olive tree. The system allows a low-pressure release of compounds.

Figure 13

Comparison traditional injection system and Invaio’s injection system (based on internal Invaio Sciences data and insights).

Figure 14

A comparison between a non-treated (on the left) and treated olive grove (on the right) located in the Otranto area (Lecce province; coordinates: Lat. 40.138095; Lon. 18.467825) within the “infected” area. It is evident to see the capability of tree restoration provided by the application of the control strategy.

Figure 15

An olive grove located in Nardò (Lecce province; coordinates: Lat. 40.187637; Lon. 17.987413) that, since 2016, has been following the control strategy described in the text. It is located in the “infected” area and borders a territory with dead or very damaged olive trees.