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.

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.

Regulation of ssb Gene Expression in Escherichia coli

Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential DNA processes such as replication, transcription, and repair. The prototypic Escherichia coli SSB protein is encoded by an ssb gene. Although the ssb gene promoters harbor an SOS box, multiple studies over several decades failed to elucidate whether ssb gene expression is inducible and SOS dependent. The SOS regulon is comprised of about 50 genes, whose transcription is coordinately induced under stress conditions. Using quantitative real-time PCR, we determined the ssb gene expression kinetics in UV- and γ-irradiated E. coli and revealed that ssb gene expression is elevated in irradiated cells in an SOS-dependent manner. Additionally, the expression of the sulA gene was determined to indicate the extent of SOS induction. In a mutant with a constitutively induced SOS regulon, the ssb gene was overexpressed in the absence of DNA damage. Furthermore, we measured ssb gene expression by droplet digital PCR during unaffected bacterial growth and revealed that ssb gene expression was equal in wild-type and SOS- bacteria, whereas sulA expression was higher in the former. This study thus reveals a complex pattern of ssb gene expression, which under stress conditions depends on the SOS regulon, whereas during normal bacterial growth it is unlinked to SOS induction. The E. coli ssb gene is SOS regulated in such a way that its basal expression is relatively high and can be increased only through stronger SOS induction. The remarkable SOS induction observed in undisturbed wild-type cells may challenge our notion of the physiological role of the SOS response in bacteria.

In vitro antifungal effect of phenylboronic and boric acid on Alternaria alternata.

The ascomycete fungus Alternaria alternata causes early blight, one of economically the most important tomato diseases. Due to frequent use of fungicides, A. alternata has developed resistance with negative economic and environmental consequences. Research of new ways to control fungal pathogens has turned its eye to environmentally friendly chemicals with low toxicity such as boronic acids. The aim of our study was therefore to test the antifungal effects of phenylboronic and boric acid in vitro on A. alternata. We isolated the pathogen from a symptomatic tomato plant and determined the minimum inhibitory concentration of phenylboronic and boric acid on A. alternata mycelial growth using the poisoned food technique. The antifungal effect was tested on a wide range of phenylboronic and boric acid concentrations (from 0.04 % to 0.3 %) applied separately to agar with mycelial disc of the pathogen. After five days of incubation, phenylboronic acid at low concentration (0.05 %) completely inhibited mycelial growth. Boric acid, in turn, did not significantly slow down mycelial growth but did reduce sporulation and confirmed its fungistatic effect. Our findings point to the potential use of phenylboronic acid to control phytopathogenic fungi. This is, to our knowledge, the first report on its antifungal effect on an agriculturally important pathogen in vitro. Moreover, since A. alternata is also a human pathogen, these results may have clinical ramifications.

Control of Early Blight Fungus (Alternaria alternata) in Tomato by Boric and Phenylboronic Acid.

Finding a suitable alternative to the small pool of existing antifungal agents is a vital task in contemporary agriculture. Therefore, intensive research has been conducted globally to uncover environmentally friendly and efficient agents that can suppress pathogens resistant to the currently used antimycotics. Here, we tested the activity of boric acid (BA) and its derivative phenylboronic acid (PBA) in controlling the early blight symptoms in tomato plants infected with pathogenic fungus Alternaria alternata. By following the appearance and intensity of the lesions on leaves of the tested plants, as well as by measuring four selected physiological factors that reflect plant health, we have shown that both BA and PBA act prophylactically on fungal infection. They did it by reducing the amount and severity of early blight symptoms, as well as by preventing deterioration of the physiological traits, occurring upon fungal inoculation. Phenylboronic acid was more efficient in suppressing the impact of A. alternata infection. Therefore, we conclude that BA, and even more so PBA, may be used as agents for controlling early blight on tomato plants, as they are both quite effective and environmentally friendly.

Phenylboronic acid as a novel agent for controlling plant pathogenic bacteria.

BACKGROUND: Phenylboronic acid (PBA) is an environmentally non-toxic substance with antimicrobial activity. Due to increasing ecological limitations in phytopharmacy and considering the development of resistance of phytopathogenic bacteria to available antibacterial agents, here we explore a possible role of PBA as an antibacterial agent of choice. RESULTS: We determined a minimal inhibitory concentration (MIC) of PBA in vitro on the Pseudomonas syringae pv. tomato (Pst) (0.5 mg/mL) and Erwinia amylovora (0.8 mg/mL), two of the most damaging plant pathogenic bacteria. In comparison, boric acid MIC was 2.5-6-fold higher than that of PBA, indicating enhanced antibacterial efficacy of the latter. Moreover, we determined the effect of PBA on cell growth and viability of both bacteria and have shown that PBA has bactericidal effect in concentrations > 1.0 mg/mL, whereas in lower concentration it is bacteriostatic. In addition, we have shown that PBA impairs Pst ability to cause symptoms on tomato plants in a dose-dependent manner, whereas solely applied PBA did not affect plant morphology at bactericidal concentrations. CONCLUSION: We report, for the first time, that PBA is a suitable agent for controlling phytopathogenic bacteria. PBA has bacteriostatic activity in lower, and bactericidal activity in higher (> 1.0 mg/mL) concentrations. When applied on tomato plants, PBA managed to suppress symptoms caused by Pst, while having no adverse effect on plants at the bactericidal concentrations. As an additional benefit, PBA is environmentally friendly. © 2022 Society of Chemical Industry.

Trends in Molecular Diagnosis and Diversity Studies for Phytosanitary Regulated Xanthomonas.

Bacteria in the genus Xanthomonas infect a wide range of crops and wild plants, with most species responsible for plant diseases that have a global economic and environmental impact on the seed, plant, and food trade. Infections by Xanthomonas spp. cause a wide variety of non-specific symptoms, making their identification difficult. The coexistence of phylogenetically close strains, but drastically different in their phenotype, poses an added challenge to diagnosis. Data on future climate change scenarios predict an increase in the severity of epidemics and a geographical expansion of pathogens, increasing pressure on plant health services. In this context, the effectiveness of integrated disease management strategies strongly depends on the availability of rapid, sensitive, and specific diagnostic methods. The accumulation of genomic information in recent years has facilitated the identification of new DNA markers, a cornerstone for the development of more sensitive and specific methods. Nevertheless, the challenges that the taxonomic complexity of this genus represents in terms of diagnosis together with the fact that within the same bacterial species, groups of strains may interact with distinct host species demonstrate that there is still a long way to go. In this review, we describe and discuss the current molecular-based methods for the diagnosis and detection of regulated Xanthomonas, taxonomic and diversity studies in Xanthomonas and genomic approaches for molecular diagnosis.

The enhancement of plant secondary metabolites content in Lactuca sativa L. by encapsulated bioactive agents.

Encapsulated bioactive agents applied to the Lactuca sativa L. present an innovative approach to stimulate the production of plant secondary metabolites increasing its nutritive value. Calcium and copper ions were encapsulated in biopolymeric microparticles (microspheres and microcapsules) either as single agents or in combination with biocontrol agents, Trichoderma viride spores, a fungal plant growth mediator. Both, calcium and copper ions are directly involved in the synthesis of plant secondary metabolites and alongside, Trichoderma viride can provide indirect stimulation and higher uptake of nutrients. All treatments with microparticles had a positive effect on the enhancement of plant secondary metabolites content in Lactuca sativa L. The highest increase of chlorophylls, antioxidant activity and phenolic was obtained by calcium-based microparticles in both, conventionally and hydroponically grown lettuces. Non-encapsulated fungus Trichoderma viride enhanced the synthesis of plant secondary metabolites only in hydroponics cultivation signifying the importance of its encapsulation. Encapsulation proved to be simple, sustainable and environmentally favorable for the production of lettuce with increased nutritional quality, which is lettuce fortified with important bioactive compounds.

Kinetics and Mechanisms of Chemical and Biological Agents Release from Biopolymeric Microcapsules.

Kinetics and mechanisms of copper cations and Trichoderma viride spores release from uncoated and chitosan coated alginate microcapsules were investigated. The gelation of a fixed amount of sodium alginate at different concentrations of copper ion solutions resulted in distinct kinetics and release mechanisms. The increase in copper cation concentration promoted, but the presence of the chitosan layer on the microcapsule surface and the increase in microcapsule size reduced the rate of active agent release. Fitting to simple Korsmeyer-Peppas empirical model revealed that the underlying release mechanism (Fickian diffusion or a combination of the diffusion and erosion mechanisms) depends on the copper cation concentration and presence of T. viride spores. The investigation pointed out that the proper selection of formulation variables helps in designing microcapsules with the desirable release of copper ions and T. viride for plant protection and nutrition.

3'-Terminated Overhangs Regulate DNA Double-Strand Break Processing in Escherichia coli.

Double-strand breaks (DSBs) are lethal DNA lesions, which are repaired by homologous recombination in Escherichia coli To study DSB processing in vivo, we induced DSBs into the E. coli chromosome by γ-irradiation and measured chromosomal degradation. We show that the DNA degradation is regulated by RecA protein concentration and its rate of association with single-stranded DNA (ssDNA). RecA decreased DNA degradation in wild-type, recB, and recD strains, indicating that it is a general phenomenon in E. coli On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the recB1080 mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3' tail (ssExos). 3'-5' ssExos deficiency is epistatic to RecA deficiency concerning DNA degradation, suggesting that bound RecA is shielding the 3' tail from degradation by 3'-5' ssExos. Since 3' tail preservation is common to all these situations, we infer that RecA polymerization constitutes a subset of mechanisms for preserving the integrity of 3' tails emanating from DSBs, along with 3' tail's massive length, or prevention of their degradation by inactivation of 3'-5' ssExos. Thus, we conclude that 3' overhangs are crucial in controlling the extent of DSB processing in E. coli This study suggests a regulatory mechanism for DSB processing in E. coli, wherein 3' tails impose a negative feedback loop on DSB processing reactions, specifically on helicase reloading onto dsDNA ends.

Encapsulation of Biological and Chemical Agents for Plant Nutrition and Protection: Chitosan/Alginate Microcapsules Loaded with Copper Cations and Trichoderma viride.

Novel chitosan/alginate microcapsules simultaneously loaded with copper cations and Trichoderma viride have been prepared and characterized. Information about the intermolecular interactions between biopolymers and bioactive agents was obtained by Fourier transform infrared spectroscopy. Encapsulation of T. viride spores and the presence of copper cations in the same compartment does not inhibit their activity. Microcapsule loading capacity and efficiency as well as swelling behavior and release depend on both the size of the microcapsule and bioactive agents. The in vitro copper cation release profile was fitted to a Korsmeyer-Peppas empirical model. Fickian diffusion was found to be a rate-controlling mechanism of release from smaller microcapsules, whereas anomalous transport kinetics controlled release from larger microcapsules. The T. viride spore release profile exhibited exponential release over the initial lag time. The results obtained opened perspectives for the future use of chitosan/alginate microcapsules simultaneously loaded with biological and chemical agents in plant nutrition and protection.

The first report on mushroom green mould disease in Croatia.

Green mould disease, caused by Trichoderma species, is a severe problem for mushroom growers worldwide, including Croatia. Trichoderma strains were isolated from green mould-affected Agaricus bisporus (button or common mushroom) compost and Pleurotus ostreatus (oyster mushroom) substrate samples collected from Croatian mushroom farms. The causal agents of green mould disease in the oyster mushroom were T. pleurotum and T. pleuroticola, similar to other countries. At the same time, the pathogen of A. bisporus was exclusively the species T. harzianum, which is different from earlier findings and indicates that the range of mushroom pathogens is widening. The temperature profiles of the isolates and their hosts overlapped, thus no range was found that would allow optimal growth of the mushrooms without mould contamination. Ferulic acid and certain phenolic compounds, such as thymol showed remarkable fungistatic effect on the Trichoderma isolates, but inhibited the host mushrooms as well. However, commercial fungicides prochloraz and carbendazim were effective agents for pest management. This is the first report on green mould disease of cultivated mushrooms in Croatia.

Gamma-irradiated RecD overproducers become permanent recB-/C- phenocopies for extrachromosomal DNA processing due to prolonged titration of RecBCD enzyme on damaged Escherichia coli chromosome.

The RecBCD enzyme of Escherichia coli consists of three subunits RecB, RecC and RecD. RecBCD enzyme activities are regulated by its interaction with recombination hotspot Chi. Biochemical and genetic evidence suggest that interaction with Chi affects RecD subunit, and that RecD polypeptide overproduction antagonizes this interaction, suggesting that intact RecD replaces a Chi-modified one. We used bacteria with fragmented chromosomes due to double-strand breaks inflicted by UV and gamma-irradiation to explore in which way increased concentrations of RecBCD's individual subunits affect DNA metabolism. We confirmed that RecD overproduction alters RecBCD-dependent DNA repair and degradation in E. coli. Also, we found that RecB and RecC overproduction did not affect these processes. To determine the basis for the effects of RecD polypeptide overproduction, we monitored activities of RecBCD enzyme on gamma-damaged chromosomal DNA and, in parallel, on lambda and T4 2 phage DNA duplexes provided at intervals. We found that gamma-irradiated wild-type bacteria became transient, and RecD overproducers permanent recB(-)/C(-) phenocopies for processing phage DNA that is provided in parallel. Since this inability of irradiated bacteria to process extrachromosomal DNA substrates coincided in both cases with ongoing degradation of chromosomal DNA, which lasted much longer in RecD overproducers, we were led to conclude that the RecB(-)/C(-) phenotype is acquired as a consequence of RecBCD enzyme titration on damaged chromosomal DNA. This conclusion was corroborated by our observation that no inhibition of RecBCD activity occurs in gamma-irradiated RecBCD overproducers. Together, these results strongly indicate that RecD overproduction prevents dissociation of RecBCD enzyme from DNA substrate and thus increases its processivity.