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For about a decade, olive groves in Apulia (Southern Italy) have been progressively destroyed by Olive Quick Decline Syndrome (OQDS), a disease caused by the bacterium Xylella fastidiosa subsp. pauca (Xfp). Recently, we described an additional wilting syndrome affecting olive trees in that area. The botryosphaeriaceous fungus Neofusicoccum mediterraneum was found associated with the diseased trees, and its high virulence toward olive trees was demonstrated. Given the common features with Branch and Twig Dieback (BTD) of olive tree, occurring in Spain and California, we suggested that the observed syndrome was BTD. During our first survey, we also found a botryosphaeriaceous species other than N. mediterraneum. In the present article, we report the morphological and molecular characterization of this fungal species which we identified as Neofusicoccum stellenboschiana. In the study, we also included for comparison additional N. stellenboschiana isolates obtained from olive trees in Latium and Tuscany region (Central Italy). The occurrence of N. stellenboschiana in olive trees is reported here for the first time in the northern hemisphere. The pathogenicity and virulence were tested in nine inoculation trials, where the Apulian N. stellenboschiana isolate was compared with the isolate from Latium and with the Apulian isolate of N. mediterraneum. Both isolates of N. stellenboschiana proved pathogenic to olive trees. They caused evident bark canker and wood discolouration when inoculated at the base of the stem of two/three-year-old trees and on one-year-old twigs. However, virulence of N. stellenboschiana was significantly lower, though still remarkable, compared with N. mediterraneum in term of necrosis progression in the bark and the wood and capacity of wilting the twigs. Virulence of N. stellenboschiana and N. mediterraneum did not substantially change when inoculations were performed in spring/summer and in autumn, suggesting that these fungal species have the potential to infect and damage olive trees in all seasons. The high thermotolerance of N. stellenboschiana was also revealed with in vitro growth and survival tests. The high virulence of these Botryosphaeriaceae species highlights their contribution in BTD aetiology and the necessity to investigate right away their diffusion and, possibly, the role of additional factors other than Xfp in the general decline of olive groves in Apulia. Hence the importance of assessing the degree of overlap of BTD/Botryosphariaceae with OQDS/Xfp is discussed.

The sharpshooter Cicadella viridis L. (Hemiptera: Cicadellidae) is the most common sharpshooter in Europe and, given its xylem feeding behavior, is considered a potential vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae). We tested X. fastidiosa subsp. pauca ST53 (Xfp) transmission capabilities of C. viridis adults, namely 1) acquisition efficiency from four host plant species—periwinkle, milkwort, lavender, alfalfa—and from two artificial diets (PD3 and Xfm), 2) inoculation efficiency to periwinkle at different times post acquisition from different plant and artificial diet sources. The main European vector species—Philaenus spumarius L. (Hemiptera: Aphrophoridae)—was used as a control. C. viridis was able to acquire Xfp from periwinkle, milkwort, and lavender, although with low efficiency (3–16%) and from artificial diets (23–25%). Successful inoculation on periwinkle was extremely rare, being observed only three times, following feeding on milkwort plant and PD3 artificial diet sources. Our study shows that C. viridis is not a relevant vector of Xfp, given the very low transmission rate in controlled conditions, and the inability to feed on olive. The low efficiency reported here correlates with ecological constraints of the vector (mainly monocots host plants, humid environments) that make it difficult to forecast a relevant role in dispersing X. fastidiosa, at least within the present distribution of the exotic bacterium in Europe. However, a possible role of this species in spreading Xf in other agroecosystems, e.g., vineyard and stone fruits grown in humid areas, cannot be excluded.

The invasive plant pathogen Xylella fastidiosa currently threatens European flora through the loss of economically and culturally important host plants. This emerging vector-borne bacterium, native to the Americas, causes several important diseases in a wide range of plants including crops, ornamentals, and trees. Previously absent from Europe, and considered a quarantine pathogen, X. fastidiosa was first detected in Apulia, Italy in 2013 associated with a devastating disease of olive trees (Olive Quick Decline Syndrome, OQDS). OQDS has led to significant economic, environmental, cultural, as well as political crises. Although the biology of X. fastidiosa diseases have been studied for over a century, there is still no information on the determinants of specificity between bacterial genotypes and host plant species, which is particularly relevant today as X. fastidiosa is expanding in the naive European landscape. We analysed the genomes of 79 X . fastidiosa samples from diseased olive trees across the affected area in Italy as well as genomes of the most genetically closely related strains from Central America. We provided insights into the ecological and evolutionary emergence of this pathogen in Italy. We first showed that the outbreak in Apulia is due to a single introduction from Central America that we estimated to have occurred in 2008 [95 % HPD: 1930–2016]. By using a combination of population genomic approaches and evolutionary genomics methods, we further identified a short list of genes that could play a major role in the adaptation of X. fastidiosa to this new environment. We finally provided experimental evidence for the adaptation of the strain to this new environment.

Xylella fastidiosa is a xylem-limited bacterium causing the Olive Quick Decline Syndrome, which is currently devastating the agricultural landscape of Southern Italy. The bacterium is injected into the xylem vessels of leaf petioles after the penetration of the insect vector’s stylet. From here, it is supposed to colonize the xylem vasculature moving against water flow inside conductive vessels. Widespread vessel clogging following the bacterial infection and causing the failure of water transport seemed not to fully supported by the recent empirical xylem anatomical observations in infected olive trees. We tested the hypothesis that the higher susceptibility to the X. fastidiosa’s infection in Cellina di Nardò compared with Leccino is associated to the higher vulnerability to air embolism of its larger vessels. Such hypothesis is motivated by the recognized ability of X. fastidiosa in degrading pit membranes and also because air embolism would possibly provide microenvironmental conditions more favourable to its more efficient aerobic metabolism. We revised the relevant literature on bacterium growth and xylem physiology, and carried out empirical field, mid-summer measurements of xylem anatomy and native embolism in olive cultivars with high (Cellina di Nardò) and low susceptibility (Leccino) to the infection by X. fastidiosa. Both cultivars had similar shoot mass traits and vessel length (~80 cm), but the highly susceptible one had larger vessels and a lower number of vessels supplying a given leaf mass. Native air embolism reduced mean xylem hydraulic conductance by ~58 % (Cellina di Nardò) and ~38 % (Leccino). The higher air-embolism vulnerability of the larger vessels in Cellina di Nardò possibly facilitates the X. fastidiosa’s infection compared to Leccino. Some important characteristics of the vector–pathogen–plant interactions still require deep investigations acknowledging both the pathogen metabolic pathways and the biophysical principles of xylem hydraulics., The olive cultivars Cellina di Nardò and Leccino have different susceptibility to the Olive Quick Decline Syndrome caused by the xylem-limited bacterium Xylella fastidiosa. The analysed infected branches of Cellina di Nardò had fewer and larger vessels, and showed higher occurrence of air embolism (higher percent loss of xylem conductance, PLC) in the field compared to those of Leccino. Results would suggest that air embolism in xylem vessels likely plays a key role in predisposing the host plant to a more virulent pathogen attack.

In this paper, a new approach for fast detection of Xylella fastidiosa bacterium symptoms on olive trees is presented. Images are taken using a multirotor unmanned aerial vehicle (UAV) equipped with a multispectral camera. A new segmentation algorithm to recognize trees is applied and images are then classified using linear discriminant analysis. It has been applied to selected sites in the Southern Italy where multispectral images of olive orchards have been acquired. The developed algorithm seems to be very promising thanks to its high mean Sorensen-Dice similarity coefficient, which demonstrates the feasibility of a correct tree individuation, and its sensitivity in detecting infected trees.

Numerous research efforts are currently underway to identify novel biocontrol agentsfor the management of diseases associated with Xylella fastidiosa. Despite the encouraging premises, most of these studies proved unable to provide long-term effects or failed once they were examined at the field-scale. An intriguing exception was that of the rhizosphere-colonizing bacterium Paraburkholderia phytofirmans PsJN, which appears efficacious to suppress Pierce’s Disease development when inoculated in grapevine infected by Xf ‘Temecula’. Having verified that PsJN did not exhibit any antagonistic in vitro effect towards Xf ‘De Donno’, we wanted to determine if it might colonize and protect olive from OQDS progress. We tested different inoculation approaches, before concluding that pin-prick method ensures the highest success rate. Motile populations of PsJN could be consistently detected distant from the inoculation point, with a newly developed SYBRÒ ­Green qPCR assay, and their long-term viability was demonstrated by successful isolation on TSA medium. We set field trials in the Apulian area affected by OQDS to investigate prophylactic and therapeutic effects of PsJN inoculation in plants naturally exposed to Xf infection. Current observations, limited to a single season, did not evidence any statistical correlation between the population dynamics of Xf and PsJN, but still, require validation on a larger time-frame. Although we collected evidence that PsJN could multiply and move in olive xylem vessels, we also observed that its concentration decreased significantly over time, suggestive of a plant response that limits PsJN expansion. We attempted a WGS metagenomic study to further investigate if PsJN might prime a host response in turn leading to a knock-on effect on the population indices of Xf and the residential microbial communities.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Xylella fastidiosa (XF) is a bacterium capable of colonizing a large number of plants, but is mainly known to cause disease in economically important crops such as citrus, grapevines, coffee and more recently olive trees. XF colonizes the xylem vessels, forming biofilm and thus blocking the passage of sap and water from roots to leaves. General symptoms related to diseases caused by these bacteria are leaves wilt, burning and subsequent leaf necrosis, leaves chlorosis and in same pathosystem plant death. The bacterium, which until recently was restricted to the American continent, in 2013 was reported in southeast Italy causing severe symptoms in olive trees leaving to dead of over one milion plants and infected eleven million. This desease was call as Olive Quick Decline Syndrome (OQDS). Since then XF has also been reported in other European countries, such as France, Spain and Portugal, infecting sweet cherry, almond, lavender and ornamental plants. Lately, in mid-2016, similar symptoms as observed in disease olive plants in Italy were also found in olive plants in the state of Minas Gerais in Brazil. The identification of XF in symptomatic olive trees in Brazil leave us to some questions about the; geographic distribution of infected plants, genetic diversity of pathogen, and natural infectivity of possible vectors involved on natural transmission. Therefore, the objetives of this work were; 1. to analyze the distribution of XF in olive orchards in southeastern Brazil; 2. to determined the genetic diversity of strains isolated from symptomatic trees; 3. to verify the natural infectivity of possible vectors. Targeting the objectives 1 and 2 aproximatley 500 samples with OQDS-like symptoms were analized for the presence of XF by using specific primers and bacterial isolation in agar medium growth following the genetic diversity studies. Those samples were sampled from twenty-five orchards located in 15 cities of states of São Paulo and Minas Gerais plus one from Espirito Santo. XF was identified in 21 of the 26 orchards analyzed. A total of 204 isolates were cultured from the symptomatic samples. Twenty-one representative strains of whole population were typed by both Multilocus Sequence type (MLST) and Simple Sequence Reapts (SSR) and the total population by the last methodology. Sixty-five percent of strains were identified as ST16 (previsouly reported in coffee plants) but others already described such as ST70 (from in hibiscus) and ST13 (from citrus) were found causing OQDS in olive plants. Beside these, new STs like the 84, 85 and 86, all belonging to subsp. pauca of X. fastidiosa were also identified in disease olive plants. Population genetic analysis using twelve SSR markers clustered the total population (204 strains) from 16 different orchards representing 12 different municipalities in only three subpopulations. Except the strains from SPA1, MG18, MGA7, MG10 and MGA4 all the remaining strains were placed in a specific subpopulation. In the SPA1 (S22°42'10.71"/W45°41'32.91") was found the largest number of private alleles and heterozygosis and also two different STs (84 and 86). The unbiased heterozygosity (HNei), ranged from 0 to 0.53 (Avg 0.20). Analyzes of possible migration routes have shown the orchards MG-A1 and MG-A2 as the largest migrant donors. X. fastidiosa was detected in at least 37% of potential vectors in commercial orchard SP-A1. A Xylella fastidiosa (XF) é uma bactéria capaz de colonizar um grande número de plantas, mas é mais conhecida por causar doenças em culturas economicamente importantes, como frutas cítricas, videiras, café e, mais recentemente, oliveiras. O XF coloniza os vasos do xilema, formando biofilme e, assim, bloqueando a passagem de seiva e água das raízes para as folhas. Os sintomas gerais relacionados com doenças causadas por estas bactérias são as folhas murchas, queimadas e subsequentemente a necrose das folhas, a clorose das folhas e, no mesmo patossistema, a morte das plantas. A bactéria, que até recentemente era restrita ao continente americano, em 2013 foi relatada no sul da Itália, causando sintomas severos em oliveiras deixando mortos de mais de um milhão de plantas e infectados onze milhões. Esta doença foi chamada de Síndrome de Declínio Rápido de Oliva (OQDS). Desde então, XF também tem sido relatado em outros países europeus, como França, Espanha e Portugal, infectando cerejeira, amendoeira, lavanda e plantas ornamentais. Ultimamente, em meados de 2016, sintomas semelhantes aos observados em plantas olivícolas na Itália também foram encontrados em olivais no estado de Minas Gerais no Brasil. Portanto, os objetivos deste trabalho foram; 1. analisar a distribuição de XF em pomares de oliva no sudeste do Brasil; 2. Determinar a diversidade genética de cepas isoladas de plantas sintomáticas; 3. verificar a infecciosidade natural de possíveis vetores. Tendo como alvo os objetivos 1 e 2, aproximadamente 500 amostras com sintomas do tipo OQDS foram analisadas quanto à presença de XF utilizando iniciadores específicos e isolamento bacteriano em meio de crescimento em ágar seguindo os estudos de diversidade genética. Essas amostras foram coletadas de 25 pomares localizados em 15 municípios dos estados de São Paulo e Minas Gerais e um do Espirito Santo. XF foi identificada em 21 dos 26 pomares analisados. Um total de 204 isolados foram cultivados a partir das amostras sintomáticas. 21 cepas representativas da população total foram diagnosticadas tanto por marcadores Multilocus Sequence Type (MLST) quanto por Simple Sequence Reapts (SSR) e a população total pela última metodologia. Sessenta e cinco por cento das cepas foram identificadas como ST16 (anteriormente relatadas em cafeeiros), mas outras já descritas, como ST70 (em hibisco) e ST13 (citros), foram encontradas causando OQDS em plantas de oliva. Além destes, novos STs como os 84, 85 e 86, todos pertencentes à subespécie. pauca de X. fastidiosa também foram identificados em plantas olivícolas. A análise genética populacional utilizando doze marcadores SSR agrupou a população total (204 cepas) de 16 pomares diferentes representando 12 municípios diferentes em apenas três subpopulações. Com excessão das amostras coletadas das localidades SPA1, MGA4, MGA10, MG18 e MGA7, todas as cepas restantes foram colocadas em apenas uma subpopulação específica. Em SP-A1 (S22 ° 42'10.71 "/ W45°41'32.91") foi encontrado o maior número de alelos privados e heterozigose e também dois diferentes STs (84 e 86). A heterozigose não-viesada de Nei (HNei) variou de 0 a 0,53 (média de 0,20). Análises de possíveis rotas migratórias mostraram os pomares MG-A1 e MG-A2 como os maiores doadores migrantes. Insetos vetores potentiais transmissores de X. fastidiosa na área SP-A1 apresentaram taxa infectividade ao redor de 37%. FAPESP: 2017/15092-9 FAPESP: 2016/02176-7

To elucidate the relationships between Xylella fastidiosa and its main European insect vector, the spittlebug Philaenus spumarius, experiments were conducted for two consecutive years to assess the kinetics, multiplication and persistence of the bacterium in the spittlebug. To this end, insects were first caged on infected source plants and then isolated, in groups of five, on olive or periwinkle plants at different times post-acquisition (3,7,14, 28, 56, and 72 days). Insects and recipient plants have been individually tested for X. fastidiosa by quantitative PCR. The bacterial load estimated in the infected insects at different times post-acquisition varied from few thousands to tens of thousands cells per insect’s head. The second experiment was carried out to assess the influence of environmental factors (temperature, season) and insect age on simulated epidemics progression on olive plants. Adults of P. spumarius, fed on infected olive branches, were then released in cages containing 16 olive plants and collected after 3-7-14-21 days. Although , most of the diagnostic tests will be carried out only in the upcoming months, preliminary results indicate a higher acquisition efficiency in September as compared to July, and a lower acquisition efficiency from periwinkle compared to olive as source plants, but higher transmission efficiency to periwinkle compared to olive as recipient plants.

Most of the diseases associated to the insect-transmitted bacterium Xylella fastidiosa (Xf) have been described in North and South America, but in the last five years widespread infections were reported in a restricted area of the Apulia region (southern Italy), on olives suffering a severe disease denoted “olive quick decline syndrome - OQDS”. Following this first report, several outbreaks have been reported in Corsica and Balearic Islands, and some in mainland France and Spain. Because many xylem sap-feeding insects can function as vectors for the transmission of this exotic pathogen in EU, several research programs are ongoing to assess the role of candidate vectors in the spread of the infections. Initial investigations identified Philaenus spumarius as the predominant vector species in the olive orchards affected by the OQDS. Additional experiments carried out over the past two years included, the spittlebugs Philaenus italosignus and Neophilaenus campestris, and of the planthopper Latilica tunetana (Hemiptera Issidae). Adults have been tested in transmission experiments to assess their ability to acquire the bacterium from infected olives and to infect different susceptible plant species. Acquisition rates determined by testing individual insects in quantitative PCR assays, ranged from 4,9% in N. campestris to 22% in P. italosignus, whereas no acquisition was recorded for L. tunetana. Successful transmissions were detected in some of the plant species exposed to P. italosignus or N. campestris, whereas L. tunetana did not transmit, even when adults were collected directly from the canopies of field-infected trees a nd tested positive in qPCR (1,4%). The ecological and epidemiological relevance of these findings are discussed. Although still limited, the data so far collected on the candidate vectors of Xf in Europe remark the major, if not exclusive, role of spittlebugs in the spread of this emerging pathogen.

LA SPERIMENTAZIONE di campo condotta nel triennio 2015-2017 in Salento ha permesso di valutare l’effi cacia di diversi insetticidi naturali e di sintesi nei confronti di Philaenus spumarius (sputacchina), principale vettore di Xylella fastidiosa dell’olivo. Le prove hanno evidenziato elevato potere abbattente e persistenza per oltre due settimane di piretroidi (deltametrina e lambda cialotrina) e neonicotinoidi (imidacloprid, tiametoxam e acetamiprid). Interessanti risultati (prove in corso) sono stati ottenuti con applicazioni a calendario di caolino su piante sane esposte per 2 anni a infezioni naturali di xylella. Si ricorda che a oggi sono registrati 1 formulato a base di acetamiprid (Epik SL) e 3 formulati a base di deltametrina (Decis Evo, Decis Easy e Decis Protect EW)., This work was partially funded by the Apulian Regional Project "Tapass» Technological Clusters and by the EU Horizon 2020-funded Projects "POnTE (Pest organisms threatening Europe, grant agreement n. 635646) and XF-Actors (Xylella fastidiosa active containment through a multidisciplinary-oriented research strategy, grant agreement n. 727987).

The meadow spittlebug Philaenus spumarius L. is a vector of the bacterium Xylella fastidiosa to a wide array of ornamental and crop species represents a serious threat to agriculture and the landscape. With the recent emergence of X. fastidiosa in Europe, particularly the strain causing Xylella-epidemics of olive trees in southern Italy, P. spumarius has been found to play a major role in the spread of the pathogen. Control of this highly polyphagous species requires an integrated pest management strategy targeting the nymphal stages and the adults. Nymphs survive on several herbaceous plants, weeds, and groundcover. Control of juvenile of P. spumarius can contribute significantly to reduce adult populations. This can be achieved through mechanical control of the weeds/groundcover; however, this may not be possible or practical to the extent necessary in many instances. Therefore, the efficacy of different insecticides, natural or inert substances (sweet orange essential oil, kaolin, and zeolite), and synthetic products (deltamethrin, buprofenzin, and imidacloprid) was tested to control the juvenile stages of P. spumarius.

The meadow spittlebug Philaenus spumarius is the first ascertained vector of X. fastidiosa in Europe, being responsible for the current Xylella-epidemics in olive orchards in Apulia (southern Italy). Preliminary studies revealed that adults acquire and transmit the bacterium after their migration from the ground vegetation to the olive canopies during the dry season (late springsummer). An effective suppression of the vector populations requires an integrated pest management strategy against nymph and adult stages. Lack of registered and authorized formulations for the control of this insect on olives, prompted for setting up field trials on comparative efficacy of different formulations. We tested the possibility of applying different insecticides, natural or inert substances (extract of citrus oil, kaolin, zeolite) and synthetic products (deltamethrin, buprofenzin, imidacloprid) to suppress the juveniles. Whereas, a total of twelve different formulations based on active compounds belonging to different chemical/ organic families and with different mechanisms of action and translocation were tested for the control of the adults. Neonicotinoids and pyrethroids showed the highest efficacy both for the control of the juvenile stages and the adult populations. The results obtained in our work provide first evidences on the efficacy of different formulations for the control of P. spumarius toward the implementation of containment strategies for X. fastidiosa in olive groves

We report here the complete and annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. pauca strain De Donno. This strain was recovered from an olive tree severely affected by olive quick decline syndrome (OQDS), a devastating olive disease associated with X. fastidiosa infections in susceptible olive cultivars.

In 2013 was detected, isolated and confirmed the bacterium Xylella fastidiosa in olive trees, oleander and almond in south-eastern Italy as the first record in the European Union. Pathogenetic tests confirmed the bacterium responsibility for a new disease: the olive quick decline syndrome (OQDS or CoDiRO). The disease is lethal, and knowledge of the candidate and actual X. fastidiosa vectors are crucial for a correct risk assessment of this threat. The xylem sap-sucking insect species present in Europe are seven sharpshooters (Cicadellidae, Cicadellinae), twenty-six spittlebugs (Aphrophoridae), seven Cercopidae, fifty-four cicadas (Cicadidae and Tibicinidae). Of these, only two species were considered potential vectors of the bacterium in Europe, Philaenus spumarius (L.) and Cicadella viridis (L.). Surveys over the infected or diseased olive groves found three homopteran species positive to X. fastidiosa. Namely, two species belonging to Aphrophoridae: P. spumarius and Neophilaenus campestris Fallén and one species of Cicadellidae: Euscelis lineolatus Brullé. Transmission tests with insects collected in infected olive groves showed the ability of P. spumarius to acquire and transmit the bacterium among olive trees and other host plants. By the time, P. spumarius is the only vector of X. fastidiosa in Europe. On-going studies will help understand the role of other candidate xylem-sap feeders vector species in the spread of the pathogenic bacterium.

Xylella fastidiosa (Xf) es una bacteria que se aloja en el xilema de las plantas afectando el flujo de agua y minerales en la savia bruta, conocida por su impacto negativo en ciertas especies comerciales de plantas causando enfermedades que afectan la producción de frutos o crecimiento de éstas, según la planta en cuestión. Xf fue aislada y descrita por primera vez en 1987 por investigadores estadounidenses. En el año 2013, se atribuyó a esta bacteria como causante del “síndrome de decaimiento rápido del olivo” (OQDS, por sus siglas en inglés) que afectó la producción de olivas en la provincia de Lecce en Italia, esto impactó no sólo la cosecha de una temporada sino que además, porque las autoridades fitosanitarias recomendaron talar y quemar los árboles afectados para no esparcir la bacteria, afectará la producción de olivas (y los subproductos derivados) durante años . En virtud de lo anterior, la Unión Europea (UE) tomó medidas particulares para contener y erradicar la bacteria en las zonas afectadas y evitar su propagación a los demás países miembros, incluyendo limitaciones a la importación de ciertas especies de plantas vivas desde ciertos países . Costa Rica se vio particularmente afectado por esta medida debido a que la palmera fénix (Phoenix roebelenii, Pr) era la principal planta ornamental exportada desde el país hacia la UE y ahora es imposible exportarla por estar incluida en dicha lista. Además, evidencia genética atribuye a la subespecie de Xf llamada pauca, presente en nuestro país, ser la variedad de Xf que afecta a los olivos. Universidad de Costa Rica UCR::Vicerrectoría de Investigación::Unidad de Gestión y Transferencia del Conocimiento para la Innovación (PROINNOVA)