Agro-active endo-therapy treated Xylella fastidiosa subsp. pauca-infected olive trees assessed by the first 1H-NMR-based metabolomic study

Abstract

Xylella fastidiosa is a xylem-limited bacterium causing a range of economically important plant diseases in hundreds of crops. Over the last decade, a severe threat due to Olive Quick Decline Syndrome (OQDS), caused by Xylella fastidiosa subspecies pauca, affected the Salento olive groves (Apulia, South-East Italy). Very few phyto-therapeutics, including a Zn/Cu citric acid biocomplex foliar treatment, were evaluated to mitigate this disease. However, the traditional foliar applications result in the agro-actives reaching only partially their target. Therefore the development of novel endo-therapeutic systems was suggested. Metabolite fingerprinting is a powerful method for monitoring both, disease progression and treatment effects on the plant metabolism, allowing biomarkers detection. We performed, for the first time, short-term monitoring of metabolic pathways reprogramming for infected Ogliarola salentina and Cima di Melfi olive trees after precision intravascular biocomplex delivery using a novel injection system. Upon endo therapy, we observed specific variations in the leaf content of some metabolites. In particular, the ¹H NMR-based metabolomics approach showed, after the injection, a significant decrease of both the disease biomarker quinic acid and mannitol with simultaneous increase of polyphenols and oleuropein related compounds in the leaf's extracts. This combined metabolomics/endo-therapeutic methodology provided useful information in the comprehension of plant physiology for future applications in OQDS control.

Figure 1

Typical aqueous ¹H NMR zgcppr spectrum of olive leaf extract. Main metabolites are indicated.

Figure 2

PCA t[1][2] scores plot (five components give R2X = 0.832, Q2 = 0.757) for leaf samples from Ogliarola salentina (circles) and Cima di Melfi (hexagons) cultivars tree at day 0 (before injection) and 0.25, 1, 2, 4, 7 and 15 days after injection. Sample symbols are colored according to different days of the treatment.

Figure 3

(a) PLS-DA t[1][2] scores plot (three components give R2X = 0.574, R2Y = 0.886; Q2 = 0.73) for leaf sample extracts from Ogliarola salentina (circles) and Cima di Melfi (hexagons) tree cultivars at day 0 (before injection), 2, and 15 after the injection. (b) Loading line plot for the model coloured according to the correlation-scaled loading (p(corr) ≥|0.5|).

Figure 4

Time course of discriminating metabolites related bucket at different intervals of 0, 0.25, 1, 2, 4, 7, and 15 days after DENTAMET injection. Mean and relative standard error refer to the average bucket intensities for each of the discriminating metabolites.

Figure 5

Stacked plot of ¹H NMR spectra of leaf sample extract at different day from the injection for Ogliarola salentina and Cima di Melfi cultivar trees. The increase of the oleuropein signals intensity was indicated.

Figure 6

Time course of copper (a) and zinc (b) leaf concentrations at different intervals (expressed as hours in the graph) of 0, 0.25, 1, 2, 4, 7, and 15 days after DENTAMET injection. The mean and relative standard error refer to the two elements' average concentrations, and the values are expressed as ppm (mg/kg of fresh weight).

Figure 7

Endotherapy device (a) An arrow-shaped metal component of the TIPS Injection System was designed to inject active ingredients precisely in the plant vascular system of olive trees. (b) Modular spray-can device of the TIPS Injection System applied to an olive tree. The system allows a low-pressure release of compounds.