The Olive Quick Decline Syndrome: A Syndemic Outbreak in the Apulia Region, Southern Italy

Soil deterioration includes both natural and man-made events, the latter usually known as “soil degradation”. This process causes a decrease of the actual or potential soil capacity to give rise to products or services. As much as 40 years ago, due to increase in salinity and alkalization, 200-300.000 ha/y were lost in industrialized countries. Twenty years later, the European Environmental Agency ¹³ listed as european soils affected by degradation (in million ha): 115 due to erosion, 42 due to wind, 85 due to acidification, 180 due to pesticide pollution, 170 due to nitrates and phosphates pollution, 33 for compaction, 3.2 for organic matter loss, 3.8 for salinization, 0.8 for waterlogging/anoxia. A few years later, aiming at contrasting progressive soil losses, the European Commission ^{14, 15} officially listed the following causes of soil degradation: erosion, pollution (localized and diffuse), salinization/alkalization, decrease of organic matter content. Currently 84% of agricultural soils in the central and southern EU are below the threshold of 3.5% of organic matter; in Italy, the average is 2% and in Apulia, in the area of OQDS, is of about 0.8-1.3%. In addition cementification, overbuilding, flooding, compaction, and loss of soil biodiversity are among other factors that depauperize the soil. The treshold of 3.5 % of organic matter is the minimum level to maintain the soil functional biodiversity, i.e. the correct functioning of biogenic cycles ¹⁶. Although the decline of organic matter content in Apulia follows the same trend as in the central and southern Europe, the damages deriving from soil erosion and salinization can be higher, particularly for the decrease of soil biodiversity and fertility ¹⁷. The survey carried out by the Competent Authorities in Apulia has shown that soil deterioration is relevant, and the ESAs (Environmentally Sensitive Areas) index, i.e. sensitivity of a given area to desertification, is highly endangered since many years throughout the whole territory from northern Tavoliere to lower Salento. This vulnerability may be due to morphological and geografical reasons as well as to anthropogenic activities, the latter leading to the loss of sustainability of the resources since more than a decade ¹⁸ (Figure 1).

Figure 1.Map of the environmental sensitivity to desertification of Puglia (ARPA, 2010). 18

The anthropogenic causes include the overuse of pesticides, the presence of abandoned pieces of land (the so-called “amateurial properties”, i.e. properties of people not resident in Apulia, visiting occasionally their property, with or without olive orchards completely overlooked and not agronomically managed), the overexploitation of water resources leading directly to progessive desertification. Although we know that this event is almost invariably irreversible, it seems that the agronomic management of the specific territory where the olive decline was started and then expanded has not changed much, mantaining therefore high the level of risk. This includes (a) the size of the olive trees, well above the average of the european olive either because of age and of absence of pruning, and (b) the overall number of olive trees, reaching about 65 million in Puglia of which 11 million only in the Salento peninsula. The agro- ecosystem has become at risk, despite the natural resilience to drought of the olive trees. Agronomists and regional technicians have begun since a decade to detect formerly undetected phytopathogens. It is well known that among the effects of desertification there is the depletion of soil life and functioning ^{19, 20}.

Throughout the Salento and coastal areas of Murgia the subterranean aquifer is of salted marine origin. Up to about thirty years ago the water wells were shallow, 3-10 meters deep. During more recent years the wells were digged through rock layer drilling to reach the deeper aquifer where salty and sweet waters co-exist, depending on the hydraulic loads of salty and sweet waters, respectively. Due to the karstic nature of the rock, along with unappropriate anthropic intervention among which numerous artesian wells, abusive (about 100.000 not registered) and overexploited, the balanced underground water resources have been compromised. Most of the surface aquifers are now empty, and their waters are called not a resource but “aquifer exudates” instead, the salty waters being recalled from the sea up to the plant roots. The risks associated with the salinization and anthropic contamination have been shown by Fidelibus and Tulipano ²¹. The same Authors predicted that the desertification of Apulia territory would have started from the Salento peninsula after 10-12 years, which then happened indeed, unfortunately. The University of Salento confirmed these serious difficulties ²². The same conclusions were drawn by the Health and Environment Centre ²³ and by the Regione Puglia ²⁴. The projects on this aspect, to mitigate the above risks, have not taken off yet. The survey of the quality of surface waters and of the aquifers ²⁵ has shown a strong chemical impairment level of water resources in the areas of the QODS, i.e. a relevant anthropic impact with poor hydro-chemical traits, including excess of nitrates and a stress above the utilization potential of water resources. It is known that prolonged drought and soil salinization close to the root canopy can lead to growth reduction of the aboveground olive organs than of the underground counterparts ²⁶, although the salt tolerance in olive may depend on the variety ²⁷, being cultivars Leccino sensitive and Frantoio or Piqual relatively tolerant. However intrusion of seawater in the aquifers can threaten soil biodiversity along with crop yields and the survival of some soil communities can be threatened, leading to the loss of biodiversity of the biota ²⁸.The same can occur for soil and rhizospheric microbes, including symbionts ^{29, 30, 31}, and their biodiversity ³². This, in turn, can expose the olive trees to the risk of disbioses and of a reduced resilience towards biotic and abiotic stresses.

Besides the risks associated to the intrusion of saline waters in the aquifers, hopefully not irreversible, in the last few decades there has been an increasing overuse of pesticides and misuse of chemical inorganic fertilizers leading to remarkable soil and aquifers stress.

The latter consist of nitrates, iron, manganese, ammonium ions, electric conducibility, clorine, sulfates which resulted almost invariably above the permitted concentrations. The causes were inferred to the misuse of nitrogen fertilizers, the overuse of zootechnical waste disposal, bad sludge management, dispersion of sewer networks, misfunctioning of disposal installations ^{23, 33}. As far as pesticides are concerned, in Apulia 11.500 tonnes of phytopharmaceuticals were used in 2013, including chlorpyrifos which was later prohibited because residues had been found in olive oil. This amount corresponds to 5.500 tonnes of active substances. In one soil sample the simultaneous presence of fifteen different pesticides was detected ³⁴. More recently, having in mind the sole biunivocal relationship OQDS/bacterium, the use of pesticides such as neonicotinoids (acetamiprid and imidacloprid) and pyretroids (deltamethrin and lambda-cyalotrin) was authorized by the national Competent Authorities to challenge the insect vector of Xylella fastidiosa, i.e. Phylaenus spumarius ^{9, 34, 35, 36}. Among herbicides, there has been an increasing usage of glyphosate as an effective aid to control weeds also in olive orchards. Although it has become the best-selling herbicide used in agriculture, horticulture, silviculture, and urban environments, it has been argued that its residues may interfere with resilience of crops to pests and phytopathogens, and with both fungal and insect biological control, which is especially relevant considering the global call for integrated pest management strategies ³⁷. A study has shown that this broad- spectrum, post-emergence herbicide alters the soil texture and microbial diversity by reducing the microbial richness and increasing the population of phytopathogenic fungi ³⁸. In temperate climate, glyphosate-based herbicides significantly decrease root mycorrhization, soil arbuscular mycorrhizal fungi (AMF) spore biomass, vesicles and propagules ^{39, 40, 41}. Studies testing glyphosate effects on AMF show an inhibition of arbuscular mycorrhizae fungal spore germination and germ tube growth or reduced mycorrhizae in soil, though only at concentrations greater than those recommended for field use. A cumulative risk assessment, never done yet, for repeated use, overusage or misuse of glyphosate in olive orchards could clarify its impact, also in connection with drainage waters, rainfalls and irrigation. In fact leaching and soil erosion by water can transport glyphosate from land to water environments ⁴², while mycorrhizae could lead to stronger absorption of glyphosate by binding and enmeshing soil particles into larger aggregates ⁴³. Leaching of glyphosate after a simulated rainfall was substantial and altered by earthworms and AMF ⁴⁴ indicating an impairment of the soil biota. An extensive utilization of glyphosate can also determine a reduction of some soil micronutrients such as zinc, copper and manganese ⁴⁵. A depletion of such ions was found in olive groves of Salento area showing severe OQDS symproms ⁴⁶.

The climatic suitability for the potential establishment of X. fastidiosa is highly variable throughout within the EU individual MSs ⁸. However, the south of the EU has been evaluated as being the most at risk. There is considerable uncertainty about recent estimates of climatic suitability, particularly considering the subspecies or sequence type level. This is due to lack of data and a potential bias in reported cases, and in particular at northern european latitudes where symptoms expression of X. fastidiosa is lacking, may bias current climatic suitability estimates. Nonetheless, variability of climatic suitability within EU individual Member States may be accounted for when designing a survey ⁴⁷, taking into consideration that the resilience and crop and olive yield may depend on climatic factors ^{48, 49}, and that the symptoms of the dessiccation can be confused with with the symptoms of water stress and drought ^{47, 50}.

During the first surveys after the occurrence of the OQDS several phytopathogens were identified in the rhizosphere, phyllosphere or as endophytic of olive trees: (i) among fungi, Phaeoacremonium aleophilum, Phaeoacremonium alvesii, Phaeoacremonium parasiticum, Phaeoacremonium italicum, Phaeoacremonium sicilianum, Phaeoacremonium scolyti, Neofusicoccum parvum, Pleurostomophora richardsiae, Pleurostomophora richardsiae ^{34, 51, 53}, Verticillium dahliae ^{2, 54}; (ii) among bacteria Xylella fastidiosa subsp. pauca ^{55, 56}. In addition Phylaenus spumarius, and other insect vectors of Xylella were found ^{57, 58}, and Zeuzera pyrina L. a Lepidopteran of the Family Cossidae; among macrobiota, nematodes were sporadically detected in olive root canopy (Figure 2). The latter, at the larval stage, start digging tiny tunnels at the apex of the smaller olive branches 1-2 years old, and then they dig larger tunnels on the main branches and finally on the trunk.

Figure 2.Symptoms of damaged olive roots by Nematodes (Pratylencus vulnus) present in Apulia.

During a subsequent survey in the buffer area, 41.520 samples were analyzed of which 1.518 symptomatic. Xylella fastidiosa was identified in only one case ⁵. Another survey showed that the bacterium could be detected, using the biochemical (ELISA) test and the molecular (PCR) reaction, in 1.8% of the tested samples ⁵⁹. A positive test is given by alive and also by dead cells of the bacterium ⁶⁰.

The above data cast some doubts on the axiom of a unique responsible of the OQDS and the consequent mitigation and curative approaches. The concurrent presence of so many and diverse mycetes in adult olive trees is by someone considered as disease aggravators ⁶¹. On the other hand the estimated probability of the most representative genera becoming infected by Xylella fastidiosa according to the current EU Xylella outbreak information places the genus Olea in the penultimate position after (in brackets the probability, 1 being equivalent to 100%): Polygala (0.551), Helichrysum (0.511), Euryops (0.471), Calicotome (0.452), Genista(0.315), Spartium (0.161), Lavandula (0.152), Cistus (0.126), Prunus (0.093), Olea (0.076), and before Vitis (0.057) ⁴⁷. All the remaining biotic and abiotic factors which can contribute to the increasing disappearence of the olive trees resilience are neglected, when designing risk mitigation measures.

Several attempts have been carried out to overcome the OQDS in Apulia, taking into consideration that the eradication of X. f. subsp. pauca and its insect vectors was not feasible and already in 2015 EFSA quoted ⁹ that other elements should be taken into account, since the establishment and spread of the pathogen have a high probability of finding a climatically suitable environment with few adverse abiotic factors. In addition the systematic use of insecticides for vector control may create environmental impacts. At that time, with regard to risk reduction options, strategies were envisaged for the prevention of introduction and for the containment of outbreaks, focusing on the two main pathways (plants for planting and infectious insect vectors) and combine the most effective options in an integrated approach. But soon it appeared that it was not enough, and that co-existance needed something more, including appropriate surveys according to different guidelines ⁴⁷. We should not forget that the severity of the outbreak is unprecedented in EU, despite the current presence of Xylella fastidiosa and its vectors in many European countries. Among the possible interventions Xiloyannis ¹⁰ enlisted the following targets (i) the content of soil organic carbon and the carbon stocks in the olive orchards, (ii) soil water retention potential, (iii) plant and soil microbiome (iv) plant management, i.e. pruning and nutrition. For the first time the dramatic situation of the olive plants underground was called into question along with a more appropriate plant management. On the other hand it is known that both crop resilience and yields depend on climatic factors ⁶², biodiversity and ability to stimulate the ecosystem functions, as well as an adequate information system of farmers ^{63, 64}. Other Aa. and International Organizations have stressed that it is essential to strenghten the binomial healthy soil - healthy and resilient plant ^{41, 65, 66}. The literature on olive microbiome is scanty. However, in a study aiming at revealing the influences on the tree microbiome but also to develop successful biocontrol strategie sagainst Verticilliumwilt in olives ⁶⁷, the olive leaf endosphere was shown to harbor mostly Proteobacteria, followed by Firmicutes, Actinobacteria, and Bacteroidetes. In addtion, a high portion of archaeal phyla Thaumarchaeota, Crenarchaeota, and Euryarchaeota were identified. All isolates with antagonistic activity against the olive-pathogenic fungus Verticillium dahliaeKleb. belong to Bacillusamyloliquefaciens.The presence of endophytes in the olive trees could be particularly relevant because it has been shown in Citrus that X. fastidiosa interacts in the xylematic channels with bacterial endophytes mainly with Methylobacteriurn mesophilicum and Curtobaterium flaccumfaciens. The latter species has been found at high frequency in asymptomatic plants, suggesting a role of this bacterium in the resistance of the plant to variegated chlorosis of agrumes caused by X. fastidiosa⁶⁸ . It is surprising that almost no data are available on the use of microbial biopesticides on olive trees as biocontrol agents for X. fastidiosa, fungi and nematodes. The same applies for the use of microbial biostimulants in olive orchards, although both categories of products are well described ⁶⁹. Preliminary results on the use of microbial consortia, containing mycorrhizal fungi and bacteria, as biofertilizers in olive orchards showing heavy symptoms of decline ⁷⁰ indicate that the treatment stimulates the microbial activity at the root canopy level and that the plant can react at the leaf canopy level on both primary and secondary branches (Figure 3). Based on these results, it can be assumed a dual activity of the microbial consortia used, a recovery of the soil vitality and the presence of endophytic microorganisms. A soil vitality recovering generally plays a key role in all crop systems, particularly for the plant growth, health and resilience against biotic and abiotic stressors. By contrast, a specific use of endophytic strains, counteracts the negative effects of pathogens, in which a potential plant-growth-promoting strain can have a certain contrast activity against endo-pathogens. Several other different experimentations on the use of microbial consortia on main crops, such as tomato, potato and mais ^{71, 72, 73} , lead to the same assumptions, indicating that the role of a certain microbial consortium is firstly the recovering of the soil vitality, as well as the presence of endophytic strains for contrasting biotic stressors. Beneficial effects were noticed also using a mixture of copper and zinc to form a hydroxyacidic complex with citric acid ⁷⁴.

Figure 3.(left) Old olive tree heavily compromised which, after the first cycle of treatments with microbial consortia, has shown a clearly improved resilience and re-vegetation – (right) Younger tree showing and improved resilience to decline after pruning and treatment with consortia containing mycorrhizal fungi and bacteria (see also https://youtu.be/AXdbb4xrImQ).

The treatment of the soil with microbial consortia as biofertilizers does not exclude the agronomic treatments on leaves, if the tree is symptomatic because of other fungi. The latter, if not entering the xylematic vessels, can be treated by pruning. Overall, the soil health and olive resilience can be improved by adopting good agronomic practices and sustainable management systems ^{75, 76}. It has been recognized that relinquishing such practices may have facilitated the extension of the OQDS ⁶¹. These practices include grafting, soil tillage, protection measures against insect vectors, pruning, fertilization with organic-based fertilizers and irrigation, soil management practices, careful monitoring and assessment of the healthy status of the environment.

There are currently 595 potential host species reported in the scientific literature ⁴⁷, 221 of which have been found infected by X. fastidiosa in natural conditions, therefore suggesting the putative endemic presence of this bacterium in the central and southern EU. According to EFSA ⁴⁷, the species Prunusdulcis, Prunus avium, Polygala myrtifolia, Spartium junceum, Nerium oleander, Rhamnus alaternus and Rosmarinus officinalis have been reported as being susceptible to at least three subspecies of X. fastidiosa. However, the decline events including the dessication in Europe is not even comparable to the ones occurring for olive trees in Apulia, the latter being even more intense in the coastal areas. Therefore, the bacterium cannot be considered the unique cause of the outbreak, although requiring by itself adequate mitigation measures. We believe that there is a need for a substantial shift of mitigation measures and of the field approach to co-existance with the multiple causes of the OQDS. In other terms we should cure the sick, not only the disease, and in this case the sick is the territory including the olive trees.

The progressive soil desertification and aquifer salinization, the progressive decrease of soil organic matter and increased (mis)use of pesticides and herbicides, the spread of phytopathogenic microorganisms including the quarantine bacterium Xylella fastidiosa subsp. pauca on commonly present host plants including the highly dense olive tree plantations, the increasing vulnerability of the territories involved in the OQDS, along with the lack of appropriate agricultural management practices and mitigation measures point clearly at a syndemic nature of the outbreak, rather than a too simplistic epidemic outbreak due to a sole, though harmful, phytopathogenic bacterium.

The syndemic nature of the threat we face in Apulia means that a more innovative and multifactorial approach is needed if we are to protect the soil health, the resilience of olive groves, the quality of the food produced and the health of the human communities.

The financial support of the Project PSR 2014- 2020 - Misura 16.2 PS-GO 2017 - Progetto AGROCIRCOLIVE – CUP ARTEA 831728 is gratefully acknowledged.