Mitigation Measures of Quick Decline Syndrome in Ancient and Monumental Olive Trees of Ostuni (Apulia, Italy) Positive to Xylella fastidiosa

1. Introduction

In previous studies the traits of OQDS (Olive Quick Decline Syndrome) in Apulia were identified as a syndemic outbreak, due to concurrent pathological events of biological, agronomic, and societal nature, requiring the development and application of risk mitigation measures [1,2,3]. Some approaches were developed, targeting mainly at one of the causes of OQDS, namely the phytopathogenic bacterium Xylella fastidiosa subsp. pauca (Xfp) [4,5]. These treatments were successfully applied to 20-year-old (cv. Ogliarola salentina) and to 60-75 years old olive trees (cv. Ogliarola salentina and Cellina di Nardò), respectively. However, it was progressively recognized that the eradication of the bacterium is impossible and that attempts to eradicate the Xfp insect vector are of limited efficacy [5,6]. Consequently, the pathogen was moved from the A1 to the A2 EPPO list [7] due to its establishment in southern Europe. Therefore, mitigation and co-existence are possibly the only effective strategies to control OQDS [8]. However, difficulties were encountered in field-testing new protocols due to stringent, and sometimes not well aligned, provisions established by the European Commission [9], European Parliament [10], Apulian Regional Authority [11,12,13], Regional Administrative Court of Bari [14], and international Authorities [6].

Here we describe an agronomic approach, applied to thirty-seven olive trees, monumental or having the monumentality traits, of the Municipality of Ostuni, aiming at (a) developing a sustainable mitigation strategy of OQDS to olive trees PCR-positive to Xylella fastidiosa, and (b) verifying at field level whether the application of the protocol allows to avoid the death or mandatory eradication measures of these trees of historical and naturalistic interest.

2. Materials and Methods

2.1. Study Location Area

Ostuni, 218 m a.s.l. and 10 Km from the Adriatic Sea, is one of the Municipalities of the Province of Brindisi (Apulia). Its surface is 22,417 Ha including 7,037 Ha cultivated with olive trees, several of them being centenary or millenary in protected areas. The soil of the Municipality is of reduced depth, with a calcareous crust on the surface, remarkably stony, and has been subjected to anthropic pressure since the last 50 years; it has high to very high salinity (750 wells are present in the territory), with progressive salinization of the aquifer [1]. The soil erosion risk assessment, according to PESERA model, is 1-3 t/Ha/year. The residents in the Municipality at the date of January 31^st, 2024 were 29,877 but thousands visitors during the year make it a very densely populated recreational area.

2.2. Olive Trees and Their Environment

The geographic coordinates of each of the 37 olive trees, among which those monumental or having traits of monumentality (cv. Cellina di Nardò, Ogliarola, Toscanina, and Cima di Melfi; Figure 1), are reported in Table 1.

The date of first sampling, i.e. at the beginning of the study, was October 21^st, 2021, for all of them. The date of second sampling, i.e. at the end of study, was October 21^st, 2024, for all of them. The 37 trees are within olive orchards, randomly spaced (Figure 2) and were managed according to the cultivation techniques of the area, i.e. no regular pruning, no soil fertilization, no control of main pests and diseases, no herbicide treatment for most of them. As a control tree we considered the one, within the same orchard, few meters from the olive ID 1255567, left without the treatment described in this study during the entire period, and being positive to Xf in 2021. Soil physicochemical characteristics of the orchards are the same as the traits reported for the Municipality. The trees were rainfed, and no irrigation was carried out. The main climatic conditions, i.e. temperature and rainfall 2021-2024 are reported in Figure 3.

2.3. Agronomic Management

The first treatment was carried out in April, 2022 by spraying 150 L/ha (per 100 L water: 500 ml industrial bleach, 400 g wettable sulphur). Five days after the treatment, both the upper canopy and the tree base were sprayed with a biostimulant microbial consortium (containing Bacillus sp., Pseudomonas sp., and Trichoderma sp.) purchased from BEA, Galazzano, Republic of San Marino. A week after the olive trees had been pollarded and grafted between the end of May and June 2022, according to the provisions established by the Regional Administrative Court of Bari [14]; a second treatment was carried out with 150 L/ha of a solution/suspension (per 100 L water: 100 g citric acid, 300 g Ergofito shark, 200 g Nemacontrol, 300 g technical urea, 200 g calcium nitrate, 100 g Ergofito boron). A third treatment was carried out in June 2023 with a solution/suspension [per 100 L water: 100 g citric acid, 250 g of ternary fertilizer 30-10-10, 300 g Ergofert start plus Bio, 200 g Nemacontrol 100 g Ergofito (Fe-Mg-Cu-Mo), 100 g Ergofito (Zn-Mn-Mg-Mo)]. The products for the second and third treatment were purchased from BEA, Galazzano, Republic of San Marino. In March 2024 the olive trees were sprayed with a solution of lime and wettable sulphur, 4-10 L per tree, followed by foliar zeolite suspension (350-400 L/ha). In June 2024 the pollards and cuts were disinfected where needed, and the tree were cleaned from occasional dry twigs.

2.4. Sampling and Analysis of Xfp by qPCR

The sampling was carried out in agreement with the guideline by EPPO [15]. After visual inspection, sampling was made on branches at least one year old and according to the cardinal points plus between cardinal points. The twigs were mainly the symptomatic ones, 6-8 for each tree, cut by using a telescopic lopper. The latter was disinfected with hypochlorite at each sampling. Each twig was placed in a sterile plastic bag, numbered according to the tree identifier, and brought to the laboratory in a refrigerated portable container within 3 hours.

The twigs from each of the plants, reported in Table 1, were collected on October 21^st, 2021, and October 21^st, 2024, respectively. The presence of Xfp was determined by Polymerase Chain Reaction (PCR) confirmed by quantitative PCR (qPCR). Samples were finely chopped and then sonicated for 1 min, then incubated 15 min at room temperature before DNA extraction following the procedure described by Dupas et al. [16]. The analysis for the presence of other phytopathogenic fungi and nematodes was planned where signs of other pathologies were present.

3. Results

3.1. Visual Inspection

A preliminary field evaluation of the same mitigation strategy (Figure 4; unpublished work) described in this study, but not included in the research here reported, encouraged us to expand the study to older olive orchards.

At the beginning of our study the symptoms of generalized OQDS were visually present in all trees, including scattered leaf scorching and twig death throughout the upper part of the canopy, or occasionally along with signs of olive tree scab (caused by Zeuzera pyrina or Pseudomonas savastanoi;Figure 5). All the trees were PCR-positive to Xf. The grafts obligatorily made according to the provisions of the Regional Authorities were dried since 2023, and only the new twigs and old branches were vegetated. After 3 years of agronomic management focusing on mitigation measures, all the trees are visually in good conditions, with normal branching and leaves, not showing symptoms of generalized OQDS, and productive. However, they were still qRT-PCR-positive to Xf subsp. pauca. An example is reported in Figure 6. The tree, left without treatments and considered a negative control, is still showing clearly the signs of OQDS in 2024 (Figure 7)

4. Discussion

The olive trees monumental or having traits of monumentality of Ostuni offered the opportunity to confirm and expand our preliminary evaluations, and the experimental approaches of other Authors, because (a) these ancient trees were the rarest affected by OQDS, (b) they had never approached to evaluate mitigation strategies, (c) they were present in the former so-called “containment area”, i.e. the area where containment legal provisions included the removal of infected plants (Regione Puglia, https://press.regione.puglia.it/-/xylella-fastidiosa-aggiornata-la-zona-di-contenimento, accessed 29.10.2024). Some recent reports are focused on the inhibition of one of the components of OQDS, namely the bacterium Xfp. Genomic analysis suggests that the pathogen arrived in southern Italy in 2008 on a coffee plant from Costa Rica (M. Saponari, 2022; see https://www.nature.com/articles/d43978-022-00008-1). It is interesting to note that previous studies indicated that the deterioration of soil properties, damage of root-knot nematodes, and accumulation of soil fungi may exacerbate the coffee plants diseases, and that the gradual decline in rhizosphere microorganism diversity and imbalanced community structure, which enriches harmful bacteria, directly contributes to coffee diseases in long-standing continuous plantations [17]. Unfortunately measures to contrast the various components of QODS in Apulian olive plantations (e.g. erratic agronomic management practices, salinization, pollution, erosion, decline of biodiversity, misuse of the territory) have not been adopted. Furthermore, to our knowledge, studies on how the olive rhizosphere and endophytic microbiome diversity and community structure can change in the presence of OQDS in Apulia are scanty. However, interest is recently growing [18,19,20,21] on the relevance of olive microbiome for the olive tree biology, resilience, and health. A pioneering work on the olive-Xf pathosystem has recently shown that in susceptible plants there is a significant change in the associated microbiota with a drastic loss of beneficial genera [22]. Studies aiming at inhibiting in vitro the growth of one component of OQDS, i.e. Xf and the application of olive tree endophytes and species of Bacillus were reviewed by Bruno and Tommasi [23]. The use of silver ultra nanoclusters [24], and the management of mineral composition of host plants have also been proposed as a control strategy of the bacterium [25]. A protocol which promotes, supports, and restores new vegetation, flowers, fruits, and oil production of the treated olive plants affected by OQDS without losing susceptible olive plants has been recently proposed [26]. In our study a co-existence and mitigation protocol was applied, based on good agronomic practices, including the treatment with microbial biostimulants. All treated trees rebuilt their foliage and new vegetation, produced drupes, and were with no or strongly reduced OQDS symptoms, resulting visually healthy, with no signs of phytopatologies, and PCR-positive to Xfp, except one (cv. Cima di Melfi, ID 1223144) which was Xfp-negative. Clearly, further investigation would be required for the trees Xfp-positive to understand whether they contain Xfp cells alive, since the PCR test only says that there is the DNA of Xfp. However, this finding confirms the observation of other Aa. that OQDS symptomatology (present in 2022 on that particular tree) can occur also in the absence of Xfp. Indeed, in the infected area of Salento, 97% of OQDS-positive trees resulted PCR-negative for Xfp; contrary to expectations, there is a not significant effect of cultivar and interaction treatment × cultivar [8]. In our case, it was interesting to note that another olive tree (outside this study, latitude 40.72638439 longitude 17.50643447), five meters from the tree ID 1255567 and negative to Xf, remained PCR-negative to Xf from 2021 to 2024, without being affected by OQDS. In the case of olive trees affected by OQDS but Xfp-negative, other phytopathologies could be associated to the olive quick decline syndrome. These phytopathologies include olive cercosporiosis, leprosy, olive wilt, olive leaf spot, peacock eye, root and crown rot, root nematodes, tree scab, verticilliosis, [27,28,29].

5. Conclusive remarks

Our field study, carried out on ancient olive trees affected or not by OQDS but positive to Xfp, indicates that the co-existence with microbial phytopathogens is possible without losing productivity or, even worst, without the elimination of the tree or forced adoption of other drastic measures such as heavy pollarding and grafting, provided that careful application of good agronomic practices is made. These findings might be relevant to those farmers who prefer to adopt alternative, more sustainable risk mitigation measures, leading to the rescue of such an important tree crop in Apulia.