Innovations towards sustainable olive crop management: a new dawn by precision agriculture including endo-therapy.

Olive trees (Olea europaea L.) are deeply interwoven with the past, present and future of Mediterranean civilizations, both as essential economical as well as cultural valuables. Olive horticulture constitutes one of the primary agroecosystems in the Mediterranean regions of today. Being inhabitant of ecological niches, Olea europaea is prone to a peculiar vulnerability towards climatic and socioeconomical transformations that are briskly reshaping regional and global agroecosystems. Because of climatic changes and the biosafety risks of global agricultural trades, olive plants are highly susceptible to newly emerging diseases and pests, and the traditional olive horticultural crop protection practices are under scrutiny towards reducing their ecological impact. Hence there is an eminent demand for a more nature-positive olive tree crop management. Recent innovations in precision agriculture are raising the prospect for innovative crop protection methods that may provide olive farmers the required agility to respond to present and future agricultural challenges. For instance, endo-therapy, which is the systemic delivery of active ingredients via trunk injection, is a technology that holds promise of a true step-change in sustainable olive crop management. Endo-therapy allows reaching vascular diseases inaccessible to foliar treatments and delivers active ingredients in a precise manner with no risks of off-target drifts. Similarly, progresses in biological precision delivery using controlled release of active ingredients based on innovative formulation technologies are showing an exciting potential for more targeted and sustainable crop protection solutions. This review summarizes the latest innovations on both physical and biological precision deliveries in the realm of olive arboriculture in the Mediterranean regions and gives an outlook how these technologies may help orchestrating innovative olive culture practices soon.

1H-NMR Metabolomics Study after Foliar and Endo-Therapy Treatments of Xylella fastidiosa subsp. pauca Infected Olive Trees: Medium Time Monitoring of Field Experiments.

Here we report the medium-term effects of foliar spray and endo-therapy treatments with different doses of a Cu/Zn citric acid biocomplex (Dentamet®) in Xylella fastidiosa infected olive trees of Salento, Apulia region (South-east Italy). Leaf extract samples from field-treated 150 years old olive trees cvs Ogliarola salentina and Cellina di Nardò were studied by 1H NMR-based metabolomics. The result of different applications of Dentamet® endo-therapy after 60, 120 and 180 days in comparison with traditional foliar spray treatment and water injection as a control have been investigated. The metabolic profile analyses, performed by 1H NMR-based metabolomic approach, indicated plant metabolites variations connected to the disease progression such as mannitol, quinic acid, and oleuropein related compounds. The best results, in terms of discrimination of the metabolic profiles with respect to water injection, were found for monthly endo-therapy treatments. Dentamet® foliar application demonstrated more specific time related progressive effectiveness with respect to intravascular treatments. Therefore, besides a possible more effective performance of endo-therapy with respect to foliar treatments, the need of further doses/frequencies trimming to obtain long-term results was also assessed. The present field studies confirmed the indication of Dentamet® effectiveness in metabolic variation induction, potentially linked with reducing the X. fastidiosa subspecies pauca related Olive Quick Decline Syndrome (OQDS) symptoms development.

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

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.

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

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.

Evaluation of seven promoters to achieve germline directed Cre-lox recombination in Arabidopsis thaliana.

Site-specific recombination systems, such as Cre-lox from bacteriophage P1, have become very important tools for plant genome engineering. In many cases a constitutive promoter is used to express the recombinase gene. However, for certain research and commercial applications constitutive Cre-mediated recombination may not be desirable. We have evaluated the potential of seven different germline promoter:cre fusions to remove a stably integrated lox cassette through Cre-mediated recombination in Arabidopsis thaliana. We monitored the functionality of each promoter in the germline of primary transformants by analyzing the presence of the recombined lox cassette in T(2) progeny. The selected germline promoters are involved in different developmental cues, including early stem cell identity (CLAVATA3), flower meristem identity (LEAFY, APETALA1), floral organ identity (AGAMOUS), and meiosis (SOLO DANCERS, DMC1, SWITCH1). For five out of these seven promoters we were able to show that efficient Cre-mediated recombination does, indeed, occur and that the recombination takes place at some point during germline development. Furthermore, a recombination efficiency of 100% is obtained when Cre-expression is regulated by the CLAVATA3 promoter. In addition, with these promoters, we observe much less variation in recombination frequency than previously reported for the 35S promoter. For these reasons, we believe that germline-specific Cre-lox recombination provides an additional tool to the site-specific recombination technology in plants.

Marker-free transgenic plants through genetically programmed auto-excision.

We present here a vector system to obtain homozygous marker-free transgenic plants without the need of extra handling and within the same time frame as compared to transformation methods in which the marker is not removed. By introducing a germline-specific auto-excision vector containing a cre recombinase gene under the control of a germline-specific promoter, transgenic plants become genetically programmed to lose the marker when its presence is no longer required (i.e. after the initial selection of primary transformants). Using promoters with different germline functionality, two modules of this genetic program were developed. In the first module, the promoter, placed upstream of the cre gene, confers CRE functionality in both the male and the female germline or in the common germline (e.g. floral meristem cells). In the second module, a promoter conferring single germline-specific CRE functionality was introduced upstream of the cre gene. Promoter sequences used in this work are derived from the APETALA1 and SOLO DANCERS genes from Arabidopsis (Arabidopsis thaliana) Columbia-0 conferring common germline and single germline functionality, respectively. Introduction of the genetic program did not reduce transformation efficiency. Marker-free homozygous progeny plants were efficiently obtained, regardless of which promoter was used. In addition, simplification of complex transgene loci was observed.