Ivan Nikolić

Taxonomy: Kingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Family Pseudomonadaceae; Genus Pseudomonas ; Species Pseudomonas syringae species complex, Genomospecies 1, Phylogroup 02b. Biology: Gram‐negative, aerobic, motile, rod‐shaped with polar flagella, oxidase negative, arginine dihydrolase negative, levan production positive, potato rot negative, tobacco hypersensitivity positive. Virulence Factors: T3SEs, toxins (syringomycin, syringopeptin, syringolin, mangotoxin, tabtoxin). Disease Symptoms: Pseudomonas syringae causes disease symptoms on beet leaves characterised as circular or irregular, dark‐edged, white to light brown necrotic spots that merge and spread over the entire leaf surface. Young leaves become completely necrotic within 7 days after the first symptom appears. Host Range: Reported disease incidence mainly occurs on beets and cucurbits in temperate regions. Greenhouse trials showed an extremely wide host range for the strains isolated from diseased beets, causing disease on plants from nine different families and 16 different plant species. Disease Control: Preventive measures focus primarily on sanitary and cultural practices. Integrated control programmes benefit from the regular application of copper formulations. The susceptibility of sugar beet cultivars to leaf spot disease varies, but no cultivar is completely resistant to the pathogen. Biological control may involve beneficial bacteria that suppress pathogens via metabolites and resource competition or by inducing systemic resistance in beet plants. Useful Websites: https://www.pseudosonseed.org/chenopods‐diseases/ ; https://www.cabi.org/isc/datasheet/44993 ; https://www.ncbi.nlm.nih.gov/assembly/?term=pseudomonas%20syringae%20pv.%20aptata ; https://www.uniprot.org/proteomes?query=pseudomonas%20syringae%20pv.%20aptata ; https://pnwhandbooks.org/plantdisease/host‐disease/beet‐red‐beta‐vulgaris‐bacterial‐leaf‐spot .

Beneficial plant-associated Bacillus species are widely explored and appreciated as eco- and environmental-friendly plant growth-promoting and biocontrol agents but often fall short of achieving the desired effect in plants. In terms of crop protection, Bacillus spp. have been repeatedly scrutinized for their secondary metabolites involved in direct antimicrobial activity towards phytopathogens or inducing systemic resistance in host plants. However, integral aspects of successful implementation of a biocontrol agent, the pronounced colonization capacity and adaptability to variations in abiotic and biotic parameters of a new environment, have been underestimated and underexplored. Here, we demonstrate that the soil-dwelling Bacillus velezensis SS-38.4 can successfully colonize the sugar beet phyllosphere, establish a stable and persistent population owing to its superior motility, ability of surfactin and biofilm production, supported by findings of genome analysis, achieving efficient suppression of the bacterial leaf spot disease. In the phyllosphere, B. velezensis SS-38.4 appears to employ an offensive strategy initiated by a swift invasion of a nonnative niche, reinforced by iron depletion and a wide arsenal of antimicrobials directed towards the phytopathogen. By displaying competitive behavior beyond its original ecological niche, B. velezensis SS-38.4 has exhibited its resilience and versatility in a challenging environment, reflecting its suitability for implementation as a biocontrol agent for efficient and comprehensive crop protection.

Disrupting quorum sensing (QS) pathways in animal and plant pathogenic bacteria is an effective strategy to mitigate infections without promoting antibiotic and pesticide resistance. This approach inhibits the production of virulence factors, biofilm formation, and toxin production, reducing bacterial pathogenicity. In plant health protection, Bacillus spp. are extensively researched and utilized as biocontrol agents; however, the potential of their AHL-lactonase-producing ability, which plays a key role as a QS inhibitor of Gram-negative pathogens, remains largely unexplored. This study examined the activity and diversity of QQ enzymes from Bacillus spp. isolates obtained from various natural sources, confirming their presence in previously unreported environments associated with agricultural fields (straw and manure). Our findings show that AiiA lactonase is the most dominant and highly conserved AHL-lactonase among Bacillus isolates from bulk soil, manure, and straw. Despite its sequence conservation, we observed significant variation in AiiA lactonase activities toward the N-hexanoyl-DL-homoserine lactone (C6-HSL) substrate. Furthermore, in silico analysis suggested that the Bacillus sp. YtnP lactonase may have a lower affinity for C6-HSL compared to AiiA lactonase. Finally, this research presents a selection of Bacillus isolates with high AiiA lactonase activity for potential testing against plant pathogens.

The rhizosphere represents a center of complex and dynamic interactions between plants and microbes, resulting in various positive effects on plant growth and development. However, less is known about the effects of indole-3-acetic acid (IAA) on aquatic plants. In this study, we report the characterization of four Pseudomonas strains isolated from the rhizosphere of the common duckweed (Lemna minor) with IAA-degradation and -utilization ability. Our results confirm previous reports on the negative effect of IAA on aquatic plants, contrary to the effect on terrestrial plants. P. putida A3-104/5 demonstrated particularly beneficial traits, as it exhibited not only IAA-degrading and -producing activity but also a positive effect on the doubling time of duckweeds in the presence of IAA, positive chemotaxis in the presence of IAA, increased tolerance to oxidative stress in the presence of IAA and increased biofilm formation related to IAA. Similarly, P. gessardii C31-106/3 significantly shortened the doubling time of duckweeds in the presence of IAA, while having a neutral effect in the absence of IAA. These traits are important in the context of plant–bacteria interactions and highlight the role of IAA as a common metabolite in these interactions, especially in aquatic environments where plants are facing unique challenges compared to their terrestrial counterparts. We conclude that IAA-degrading and -producing strains presented in this study might regulate IAA effects on aquatic plants and confer evolutionary benefits under adverse conditions (e.g., under oxidative stress, excess of IAA or nutrient scarcity).

Pseudomonas syringae pv. aptata is a member of the sugar beet pathobiome and the causative agent of leaf spot disease. Like many pathogenic bacteria, P. syringae relies on the secretion of toxins, which manipulate host-pathogen interactions, to establish and maintain an infection. This study analyzes the secretome of six pathogenic P. syringae pv. aptata strains with different defined virulence capacities in order to identify common and strain-specific features, and correlate the secretome with disease outcome. All strains show a high type III secretion system (T3SS) and type VI secretion system (T6SS) activity under apoplast-like conditions mimicking the infection. Surprisingly, we found that low pathogenic strains show a higher secretion of most T3SS substrates, whereas a distinct subgroup of four effectors was exclusively secreted in medium and high pathogenic strains. Similarly, we detected two T6SS secretion patterns: while one set of proteins was highly secreted in all strains, another subset consisting of known T6SS substrates and previously uncharacterized proteins was exclusively secreted in medium and high virulence strains. Taken together, our data show that P. syringae pathogenicity is correlated with the repertoire and fine-tuning of effector secretion and indicate distinct strategies for establishing virulence of P. syringae pv. aptata in plants.

The diversity of plant-associated bacteria is vast and can be determined by 16S rRNA gene metabarcoding. Fewer of them have plant-beneficial properties. To harness their benefits for plants, we must isolate them. This study aimed to check whether 16S rRNA gene metabarcoding has predictive power in identifying the majority of known bacteria with plant-beneficial traits that can be isolated from the sugar beet (Beta vulgaris L.) microbiome. Rhizosphere and phyllosphere samples collected during one season at different stages of plant development were analyzed. Bacteria were isolated on rich unselective media and plant-based media enriched with sugar beet leaves or rhizosphere extracts. The isolates were identified by sequencing the 16S rRNA gene and tested in vitro for their plant-beneficial properties (stimulation of germination; exopolysaccharide, siderophore, and HCN production; phosphate solubilization; and activity against sugar beet pathogens). The highest number of co-occurring beneficial traits was eight, found in isolates of five species: Acinetobacter calcoaceticus, Bacillus australimaris, B. pumilus, Enterobacter ludwiigi, and Pantoea ananatis. These species were not detected by metabarcoding and have not previously been described as plant-beneficial inhabitants of sugar beets. Thus, our findings point out the necessity of a culture-dependent microbiome analysis and advocate for low-nutrient plant-based media for high-yield isolation of plant-beneficial taxa with multiple beneficial traits. A culture-dependent and -independent approach is required for community diversity assessment. Still, isolation on plant-based media is the best approach to select isolates for potential use as biofertilizers and biopesticides in sugar beet cultivation.

Plant pathogenic strains of Pseudomonas syringae (Psy) spp. have been detected in nonagricultural habitats, including those associated with the water cycle. Their presence in aquatic systems allows dissemination over long distances, especially with irrigation practices. In May 2021, we sampled 15 sites along the Danube River Basin in Serbia to gain insight into P. syringae abundance and diversity. We identified 79 Psy strains using Psy-specific primers, and a partial sequence of the citrate synthase (cts) house-keeping gene has served for phylogenetic diversity assessments. Phenotypic diversity determination included characterizing features linked with survival and pathogenic lifestyle. The ice nucleation activity, pectinolytic activity, swimming and swarming assays, and hypersensitive reaction on plants were tested. Psy was detected at ten of 15 sites examined at abundance ranging from 1.0 × 10² to 1.2 × 10⁴ CFU/L. We discovered the presence of four phylogroups, with phylogroup 2 being the most abundant, followed by phylogroups 7, 9, and 13. The hypersensitive reaction was induced by 68.63% of the isolates from the collection. A partial sequence comparison of the cts gene showed 100% similarity between isolates from cherry plants epidemics in Serbia caused by Psy and isolates from the Danube River. Our results suggest that the Danube River, extensively used for irrigation of agricultural fields, harbors diverse strains of Psy, which possess various features that could lead to potential disease outbreaks on crops. This study represents the first in-depth analysis of Psy abundance and diversity in the Danube River Basin. It sets the ground for future pre-epidemic studies and seasonal monitoring of Psy population dynamics.