A study on seasonal abundance of Auchenorrhyncha species and their infectivity by Xylella fastidiosa in the Apulia region of Italy was conducted to identify ideal periods for monitoring and adoption of potential control measures against insect vectors. Adult populations of Auchenorrhyncha species were monitored monthly over a 2-yr period from five olive groves. A total of 15 species were captured, identified, and tested for presence of X. fastidiosa by polymerase chain reaction (PCR). For three species, Philaenus spumarius L., Neophilaenus campestris (Fallèn), and Euscelis lineolatus Brullé, positive reactions to X. fastidiosa were obtained, on average, in 16.3, 15.9 and 18.4% of adult insects, respectively. Philaneous spumarius was the dominant species (39.8% of total Auchenorrhyncha captured) with the highest adult abundance in summer months. Adult P. spumarius and N. campestris were first detected between March and May in both years, and all insects tested during these periods (year 1: n = 42, year 2: n = 132) gave negative reactions to X. fastidiosa by PCR. Similarly, first adults of E. lineolatus that appeared from October to November (year 1: n = 20, year 2: n = 15) tested negative for presence of X. fastidiosa . Given the lack of transstadial and transovarial transmission of X. fastidiosa and considering that P. spumarius is univoltine, control measures against nymphal stages of P. spumarius should be investigated as means of population suppression to reduce spread of X. fastidiosa in olive groves.

Olive quick decline syndrome (OQDS), which is caused by Xylella fastidiosa, poses a severe threat to the agriculture of Mediterranean countries and causes severe damage to the olive trees in Italy. Since no effective control measures are currently available, the objective of this study was the screening of antagonistic bacteria that are potentially deployable as biocontrol agents against X. fastidiosa. Therefore, two approaches were used, i.e., the evaluation of the antagonistic activity of (i) endophytic bacteria isolated from two different cultivars of olive trees (Leccino and Ogliarola salentina) and (ii) epiphytic bacteria isolated from the phyllospheres of different host plant species of X. fastidiosa. In vitro dual culture tests showed that 12 out of 200 isolates inhibited X. fastidiosa growth, with appearances of clear zones between 4.0 and 38.6 mm. 16S rRNA gene sequencing revealed different species of the genera Paenibacillus, Bacillus, Pantoea, Microbacterium, Stenotrophomonas, Delftia, and Pseudomonas. Furthermore, an investigation for antimicrobial activity identified 5 out of the 12 antagonistic bacteria, Paenibacillus rigui, Bacillus subtilis, Bacillus pumilus, Microbacterium oxydans, and Stenotrophomonas rhizophila, that were able to produce culture filtrates with inhibitory activities. Our results are promising for further investigation to develop an eco-sustainable strategy to control X. fastidiosa using biocontrol agents or their secreted metabolites.

Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations.

The high level of copper (Cu) accumulation in the soil, the risk of surface water contamination, and the potential public health problems due to Cu entering the food chain have raised concerns on the use of Cu compounds in agriculture, including olive growing. As a consequence, there is worldwide regulatory pressure on agricultural systems to limit the use of Cu compounds. Hence, a field trial was carried out to evaluate the effectiveness of low copper containing chemicals to control olive leaf spot (OLS) disease caused by Venturia oleaginea. The trial was conducted in 2021 in an olive (cv. Nabali Baladi) grove in Palestine. Copper complexed with lignosulphonate and gluconate (Disper Cu Max^®) and the self-defense inducer Disper Broton GS^® were evaluated and compared to dodine and the traditionally and frequently used copper hydroxide. In addition, untreated trees were used as the control. Treatments were made in March, July, and August. In March 2021, leaves grown in 2020 were present and 100% infected. V. oleaginea infections caused defoliation in untreated and treated olive trees with varying degrees of intensity: the Control had the most defoliation, followed by copper hydroxide and Disper Cu Max^®, whereas dodine and, in particular, Disper Broton GS^® had the least. All treatments reduced symptomatic leaves but their efficacy varied significantly: copper hydroxide was the least effective, Disper Cu Max^® was intermediate, dodine and, mainly, Disper Broton GS^® were the most effective. Overall, the results are promising since Disper Cu Max^® and Disper Broton GS^® were able to significantly reduce OLS damage and the amount of copper used for treatments.

Endophytes are symptomless fungal and/or bacterial microorganisms found in almost all living plant species. The symbiotic association with their host plants by colonizing the internal tissues has endowed them as a valuable tool to suppress diseases, stimulate growth, and promote stress resistance. In this context, the study of culturable endophytes residing the sapwood of Apulian olives might be a promising control strategy for xylem colonizing pathogens as Xylella fastidiosa. To date, olive sapwood cultivable endophytes are still under exploration; therefore, this work pursues a study of cultivable endophytes occurrence variation in the sapwood of different olive varieties under the effect of seasonality, geographical coordinates, and X. fastidiosa infection status. Our study confirms the stability of sapwood endophytic culturable communities in the resistant olive variety and presents the seasonal and geographical fluctuation of olive trees’ sapwood endophytes. It also describes the diversity and occurrence frequency of fungal and bacterial genera, and finally retrieves some of the sapwood-inhabiting fungal and bacterial isolates, known as biocontrol agents of plant pathogens. Thus, the potential role of these bacterial and fungal isolates in conferring olive tree protection against X. fastidiosa should be further investigated.

Early detection of Xylella fastidiosa outbreaks in Apulian olive groves is crucial, especially in buffer zones and pathogen-free areas where olive trees are asymptomatic. Three studies were conducted. Two were on the spatial and temporal progression of X. fastidiosa infections in tree canopies of asymptomatic or mildly symptomatic olive trees of tolerant (‘Leccino’) and susceptible (‘Cellina di Nardò’ and ‘Ogliarola salentina’) cultivars. Despite different trends in pathogen infection rates and concentrations between ‘Leccino’ and susceptible olive cultivars over the study period, results showed that sampling was most effective in the mid-upper part of tree canopies throughout the year, excluding the warmest and coldest periods. Stem xylem tissues were the most appropriate for detecting the pathogen compared to lower parts of mature leaves with petioles, using serological and molecular assays. Based on these results, a third study was conducted to compare molecular and serological tests (qPCR, real-time LAMP, DAS-ELISA, DTBIA) for detection of X. fastidiosa in the mid-upper part of asymptomatic branches of infected ‘Leccino’ trees that were sampled in an appropriate collection time, using stem xylem tissue as the most appropriate matrix for testing. The molecular methods showed the greatest sensitivity, with no undetermined results, while among the serological assays, DTBIA was more sensitive than DAS-ELISA. An improved protocol for monitoring asymptomatic olive trees is recommended.