RobHortic is a remote-controlled field robot that has been developed for inspecting the presence of pests and diseases in horticultural crops using proximal sensing. The robot is equipped with colour, multispectral, and hyperspectral (400–1000 nm) cameras, located looking at the ground (towards the plants). To prevent the negative influence of direct sunlight, the scene was illuminated by four halogen lamps and protected from natural light using a tarp. A GNSS (Global Navigation Satellite System) was used to geolocate the images of the field. All sensors were connected to an on-board industrial computer. The software developed specifically for this application captured the signal from an encoder, which was connected to the motor, to synchronise the acquisition of the images with the advance of the robot. Upon receiving the signal, the cameras are triggered, and the captured images are stored along with the GNSS data. The robot has been developed and tested over three campaigns in carrot fields for the detection of plants infected with ‘Candidatus Liberibacter solanacearum’. The first two years were spent creating and tuning the robot and sensors, and data capture and geolocation were tested. In the third year, tests were carried out to detect asymptomatic infected plants. As a reference, plants were analysed by molecular analysis using a specific real-time Polymerase Chain Reaction (PCR), to determine the presence of the target bacterium and compare the results with the data obtained by the robot. Both laboratory and field tests were done. The highest match was obtained using Partial Least Squares-Discriminant Analysis PLS-DA, with a 66.4% detection rate for images obtained in the laboratory and 59.8% for images obtained in the field.

A protocol for the specific detection and quantification of ‘ Candidatus Liberibacter solanacearum’ in carrot seeds using real‐time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘ Ca . L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter‐like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post‐germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect‐proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘ Ca . L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘ Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real‐time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘ Ca . L. solanacearum’ in seed lots is required.

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5–92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.

In 2017, Xylella fastidiosa, a quarantine plant-pathogenic bacterium in Europe, was detected in almond trees associated with leaf scorch symptoms in Alicante, a Mediterranean area in southeastern mainland Spain. The bacterium was detected by serological and molecular techniques, isolated in axenic culture from diseased almond trees, and identified as X. fastidiosa subsp. multiplex sequence type (ST) 6. Inoculation experiments on almond plants in greenhouse trials with a characterized strain of X. fastidiosa subsp. multiplex ST6 isolated in the outbreak area have proved that it was able to multiply and systemically colonize inoculated plants. Disease symptoms characteristic of leaf scorch like those observed in the field were observed in the inoculated almond trees after 1 year. Furthermore, the pathogen was reisolated and identified by molecular tests. With the fulfillment of Koch’s postulates, we have demonstrated that X. fastidiosa is the causal agent of the almond leaf scorch disease in the Alicante outbreak.

The genus Erwinia includes plant-associated pathogenic and non-pathogenic species. Among them, all species pathogenic to pome fruit trees (E. amylovora, E. pyrifoliae, E. piriflorinigrans, Erwinia sp. from Japan) cause similar symptoms, but differ in their degrees of aggressiveness, i.e. in symptoms, host range or both. The presence of plasmids of similar size, in the range of 30 kb, is a common characteristic that they possess. Besides, they share some genetic content with high homology in several genes associated with exopolysaccharide production and hence, with virulence, as well as in some other genes. Knowledge of the content of these plasmids and comparative genetic analyses may provide interesting new clues to understanding the origin and evolution of these pathogens and the level of symptoms they produce. Furthermore, genetic similarities observed among some of the plasmids (and genomes) from the above indicated pathogenic species and E. tasmaniensis or E. billingiae, which are epiphytic on the same hosts, may reveal associations that could expose the mechanisms of origin of pathogens. A summary of the current information on their plasmids and the relationships among them is presented here.

Huanglongbing (HLB) is the most devastating citrus disease and is associated with three bacterial species of the genus ‘Candidatus Liberibacter’ transmitted by insect vectors. The early detection of HLB is based on PCR methods, and it is one of the cornerstones for preventing incursion into disease-free countries. However, the detection of phytopathogenic bacteria with PCR-based methods is problematic in surveys that include a variety of samples of different origins. Here, we first report the proportion of amplifications obtained by two standardized real-time PCR methods for the diagnosis of HLB in various environmental samples that include plants, psyllid vectors, and parasitic wasps of the psyllids. The results of 4915 samples showed that 9.3% of them were amplified by the first rapid screening test and only 0.3% by the more specific tests. Most of the amplifications were associated with parasitic wasps. We designed the primers external to the target regions of both real-time PCR protocols to determine if amplifications belonged to one of three ‘Ca. Liberibacter’ species associated with HLB. The bioinformatic analysis of the sequences obtained with these primers revealed that all these amplifications came from the presence of other prokaryotic organisms in the samples. The primers developed in this study overcome the problem of undesired amplification in environmental samples. Thus, they could be used in future survey protocols to prevent the eradication of negative trees and the generation of unjustified alarms.