Varietal Susceptibility of Olive to Pseudomonas savastanoi pv. savastanoi and the Antibacterial Potential of Plant-Based Agents.

Olive knot disease, caused by the bacterium Pseudomonas savastanoi pv. savastanoi, causes great damage in olive orchards. While control measures of P. savastanoi pv. savastanoi in olive orchards primarily rely on pruning and copper-based treatments, the use of antibiotics as bactericidal preparations in agriculture is limited and highly restricted. However, plants are naturally endowed with protective molecules, such as phenolic compounds, which defend them against herbivores, insects, and microorganisms. This research aimed to test the virulence of five strains of P. savastanoi pv. savastanoi isolated from different growing regions and olive varieties, and to examine whether there is a difference in plant susceptibility based on the variety. An additional goal was to test the antimicrobial activity of olive mill wastewater, known for its high content of phenolic compounds, and aqueous garlic hydrolysate, as well as to compare them with a commercial copper-based product, pure hydroxytyrosol, and a standard antibiotic as references. Analysis of knot characteristics showed variations in the virulence of the P. savastanoi pv. savastanoi strains, with the highest virulence being observed for the strain I7L and the lowest virulence for the strain B45C-PR. The olive cultivar Rosinjola displayed higher susceptibility compared to Frantoio, Buza, and Leccino, while cv. Istarska bjelica exhibited the least susceptibility compared to the other investigated olive cultivars. In an attempt to explore alternative solutions for disease control, in vitro tests revealed that the phenol HTyr, GE, and the wastewater with the highest total phenolic content (cv. Istarska bjelica) possess the highest antibacterial activity. This supports the role of polyphenols in host defense, aligning with previous field observations of lower susceptibility of cv. Istarska bjelica to olive knot disease. These findings highlight the complex nature of olive knot interactions with bacterial strains and olive cultivars, simultaneously accentuating and underscoring the importance of considering the host's defenses as well as bacterial virulence in disease management strategies.

Correction: Haegeman et al. Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests. Plants 2023, 12, 2139.

In the original publication [...].

The Antibacterial Effect of Selected Essential Oils and Their Bioactive Constituents on Pseudomonas savastanoi pv. savastanoi: Phytotoxic Properties and Potential for Future Olive Disease Control.

Plant pathogenic bacteria pose a significant threat to olive cultivation, leading to substantial economic losses and reduced yield. The efficacy of antimicrobial agents against these pathogens is of great interest for sustainable disease management strategies. As such, the management of olive knot disease is one of the major challenges in olive protection. In the presented study, through a series of in vitro assays, we investigated the antimicrobial effect of six essential oils (EOs) and their most concentrated constituents against causative agent of olive knot disease-Pseudomonas savastanoi pv. savastanoi, highlighting the high potential of Origanum compactum EO and its constituent carvacrol. Carvacrol exhibited the highest potential for practical application, demonstrating membrane disruption as its mechanism of action even at the lowest concentration. The bactericidal effect of antimicrobials was confirmed in a time-kill assay, where concentrations of MIC, 2× MIC, and 4× MIC were evaluated. Some of the applied treatments resulted in inhibition equal or higher than copper-based treatment. Additionally, we assessed the phytotoxicity of carvacrol by foliar application on olive cv. Leccino. The appearance of phytotoxic injuries majorly occurred on the young leaves of olive plants, with the highest proportion of damaged canopy observed when the 2× MIC concentration was applied. Due to its great efficiency against P. savastanoi pv. savastanoi in vitro, these findings highlight the potential of carvacrol as a molecule of interest for the development of environmentally friendly biopesticides. This study also contributes to the advancement of disease management practices in olive cultivation, leading to enhanced crop protection.

Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests.

High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well.

Identification and Characterisation of Pseudomonas savastanoi pv. savastanoi as the Causal Agent of Olive Knot Disease in Croatian, Slovenian and Portuguese Olive (Olea europaea L.) Orchards.

Strains of Pseudomonas savastanoi pv. savastanoi (Pss), isolated from infected olive trees (Olea europaea L.) in three European countries (Croatia, Slovenia and Portugal) were identified and characterised according to their colony morphology, physiological and biochemical features. According to the LOPAT scheme, 38.6% of Pss isolates were grouped in the Ib cluster. The Portuguese Pss strains were fully consistent with the typical LOPAT profile for this bacterium. Conversely, most Slovenian Pss strains showed delayed oxidase activity, whilst Croatian Pss strains did not produce any fluorescent pigment when grown in vitro. For Pss molecular identification, both end-point and real-time PCR were used, as well as MALDI-TOF, which was additionally used for proteomic analysis and the subsequent species identification of a number of strains that showed deviations from expected LOPAT results. Pss was confirmed as a causal agent of olive knot disease in 46.6% of olive orchards screened. Overall, these data suggests a possible correlation of certain Pss features with the geographical origin and the ecological niche of Pss isolates.

Identification and Characterization of 33 Bacillus cereus sensu lato Isolates from Agricultural Fields from Eleven Widely Distributed Countries by Whole Genome Sequencing.

The phylogeny, identification, and characterization of 33 B. cereus sensu lato isolates originating from 17 agricultural soils from 11 countries were analyzed on the basis of whole genome sequencing. Phylogenetic analyses revealed all isolates are divided into six groups, which follows the generally accepted phylogenetic division of B. cereus sensu lato isolates. Four different identification methods resulted in a variation in the identity of the isolates, as none of the isolates were identified as the same species by all four methods-only the recent identification method proposed directly reflected the phylogeny of the isolates. This points to the importance of describing the basis and method used for the identification. The presence and percent identity of the protein product of 19 genes potentially involved in pathogenicity divided the 33 isolates into groups corresponding to phylogenetic division of the isolates. This suggests that different pathotypes exist and that it is possible to differentiate between them by comparing the percent identity of proteins potentially involved in pathogenicity. This also reveals that a basic link between phylogeny and pathogenicity is likely to exist. The geographical distribution of the isolates is not random: they are distributed in relation to their division into the six phylogenetic groups, which again relates to different ecotypes with different temperature growth ranges. This means that we find it easier to analyze and understand the results obtained from the 33 B. cereus sensu lato isolates in a phylogenetic, patho-type and ecotype-oriented context, than in a context based on uncertain identification at the species level.

Molecular Characterization of Divergent Closterovirus Isolates Infecting Ribes Species.

Five isolates of a new member of the family Closteroviridae, tentatively named blackcurrant leafroll-associated virus 1 (BcLRaV-1), were identified in the currant. The 17-kb-long genome codes for 10 putative proteins. The replication-associated polyprotein has several functional domains, including papain-like proteases, methyltransferase, Zemlya, helicase, and RNA-dependent RNA polymerase. Additional open reading frames code for a small protein predicted to integrate into the host cell wall, a heat-shock protein 70 homolog, a heat-shock protein 90 homolog, two coat proteins, and three proteins of unknown functions. Phylogenetic analysis showed that BcLRaV-1 is related to members of the genus Closterovirus, whereas recombination analysis provided evidence of intraspecies recombination.

Agrobacteria Enhance Plant Defense Against Root-Knot Nematodes on Tomato.

The increased incidence of the crown gall disease caused by Agrobacterium tumefaciens has long been associated with activities of root-knot nematodes (Meloidogyne spp.). Pot experiments on tomato were designed to assess plant vitality, nematode reproduction, and crown gall incidence in combined infection with Agrobacterium and Meloidogyne spp. on tomato roots. Results suggest that tomato plants infected with pathogenic A. tumefaciens 2 days before the nematodes show enhanced plant defense against M. ethiopica resulting in lower egg and gall counts on roots 45 and 90 days postinoculation (dpi); no significantly enhanced defense was observed when the plant was inoculated with bacteria and nematodes at the same time. Split-root experiments also showed that the observed interaction was systemic. Reverse-transcription quantitative polymerase chain reaction analysis that targeted several genes under plant hormonal control suggests that the suppression was mediated via systemic acquired resistance by the pathogenesis-related protein 1 and that M. ethiopica did not enhance the defense reaction of tomato against Agrobacterium spp. Nematodes completely inhibited tumor growth in a 45-day experiment if inoculated onto the roots before the pathogenic bacteria. We conclude that the observed antagonism in the tested pathosystem was the result of initially strong plant defense that was later suppressed by the invading pathogen and pest.