In this work by encapsulation technique we have synthetized three new clove bud essential oil (CEO) Emulsifiable Concentrate (EC) formulations depending on the carrier (synthetic zeolite- F-CSZ, nature zeolite- F-CNZ and gelatin- F-CG). The main idea was to develop an eco-friendly biopesticide that can find use in plant protection as an alternative to the use of conventional pesticides. By encapsulation we wanted to enable water solubility and ensure prolonged efficacy of the essential oil. Biological activity of designed CEO formulations was tested on potato tuber moth Phthorimaea operculella (fumigant mode of action), gray mold fungal pathogen Botrytis cinerea (preserver coatings), and soft rotting bacterial pathogens Pectobacterium carotovorum (subsp. carotovorum and brasiliensis) and Dickeya dianthicola (direct competition). CEO formulations evinced a prolonged action on mortality of P. operculella during the insects’ exposure to the concentration of the emulsions of 40 µL L⁻¹ air. The mortality gradually decreased from a probability of 100% after the first 24 h to 50% after 5 days for F-CSZ or after 4 days for F-CNZ and F-CG. The most promising formulation is F-CSZ enabling activity during 14 days of exposure, while the effect of the other two formulations lasted 10 days. All three formulations produced a strong fungicidal effect against B. cinerea by preventing infection and disease development. The best efficacy was evidenced with F-CSZ (synthetic zeolite as a carrier) showing 100% efficacy when it was used even at the lowest tested concentration of active CEO (1%). The results of in vitro testing against soft rot pathogens determined the MIC value of CEO formulations to be 1% of active CEO. By this research, we present a novel perspective on the use of essential oils as an alternative, environmental biopesticide. CEO formulations can be commercially exploited as a fumigant or preserver coatings to extend the shelf life of stored products or the fresh-fruit market.

Unusual blight-like symptoms appeared on highbush blueberry plants in Serbia during August 2015 and infected plants showed browning and reddening of leaves, drying of foliage and brown discolouration of internal vascular stem tissues. The objective of this study was to isolate and confirm a causal agent of the disease. Five diseased blueberry plants (2-year-old), with visible brown discolouration in the wood, were collected for isolation on potato dextrose agar (PDA). Morphological analysis of the selected fungal isolates showed the presence of abundant black, round to oblong, or irregularly shaped microsclerotia immersed in the PDA. Dark, globose pycnidia formed on water agar with an initially hyaline, granular content and single-celled conidia, indicating the presence of plant pathogenic fungus Macrophomina phaseolina associated with symptomatic plant tissues. Pathogenicity was confirmed on potted blueberry plants based on the initial symptoms of leaves turning yellowish to brown at the leaf edges, followed by the defoliation of leaves of the inoculated stems. Discolouration of vascular tissues was also observed on transverse sections of inoculated stems. The pathogen M. phaseolina was confirmed using molecular analysis of the ITS1-5.8S-ITS2 region of rDNA and a part of the TEF-1α gene region. This is the first report of M. phaseolina causing a blight disease on highbush blueberry in Serbia. The study should help in elucidating disease symptomatology and provide information on the risk which this fungus could pose in blueberry production.

Blackleg outbreaks were noticed on three fields (about 100 ha total) in 2 consecutive years (2018, 2019) in one of the main potato growing areas in Serbia (Bačka region, Vojvodina). The percentage of infected plants reached 40 to 70%, with 10.5 to 44.7% yield reductions. From the three fields, out of 90 samples Pectobacterium carotovorum subsp. brasiliensis was most frequently identified and diagnosed as causal agent of potato blackleg in Serbia for the first time (29 isolates). Dickeya dianthicola was a less frequently causative bacterium, which was also noticed for the first time (nine isolates). A total of 38 isolates were characterized based on their phenotypic and genetic features, including a pathogenicity test on potato. The repetitive element PCR (rep-PCR) using BOX, REP, and ERIC primer pairs differentiated five genetic profiles among 38 tested isolates. Multilocus sequence analysis (MLSA) of four housekeeping genes, acnA, gapA, icdA, and mdh, revealed the presence of three so far unknown P. c. subsp. brasiliensis multilocus genotypes and confirmed clustering into two main genetic clades as determined in other studies. MLSA also revealed the presence of a new genotype of D. dianthicola in Serbia.

Potato blackleg is frequently observed on the production fields in the Bačka region of Vojvodina province, which is one of the largest potato-growing areas in Serbia. This disease usually occurs during June and July. In July 2020, blackleg symptoms in the form of stem necrotic lesions, vascular discoloration, hollow stems, and wilting of whole plants were noted on potato cultivar VR808 on a field 28 ha in size located in Maglić village (GPS coordinates 45.349325 N, 19.542768 E). Disease incidence was estimated at 20-25%. Isolations were performed from 12 potato samples on Crystal Violet Pectate medium (CVP). Stem sections consisted of brown lesions and healthy tissue (c.10 cm) were surface sterilized with ethyl alcohol 70% (w/v) and rinsed with sterile distilled water. Small pieces of tissue were taken at the edges of stem lesions (between healthy and diseased tissue) were soaked in phosphate buffer saline for 20 min and plated using a standard procedure (Klement et al. 1990). Single colonies that formed pits after 48 hours at 26 °C were re-streaked onto Nutrient Agar (NA) where creamy white colonies with smooth surfaces were formed. A total of 30 isolates were selected and DNA isolated from the colonies was further analyzed by polymerase chain reaction (PCR) using the partial dnaX gene (DNA polymerase subunit III gamma/tau) with primer pair dnaXf/dnaXr for Pectobacterium and Dickeya species identification (Slawiak et al. 2009). A single characteristic band of 535 bp was amplified in all isolates (Slawiak et al. 2009). DNA sequence alignment showed two distinct groups of isolates (Fig.S1), which were genetically uniform within each group. Using BLASTn search, it was established that the dnaX sequence of the first group (consisting of 19 Serbian potato isolates) had 99.79% identity with NCBI-deposited Pectobacterium versatile strains 14A and 3-2 from potato from Belarus (Acc. No. CP034276 and CP024842, respectively) as well as SCC1 from Finland (Acc. No. CP021894). The remaining 11 dnaX sequences had 100% identity with Pectobacterium carotovorum subsp. carotovorum strain CFBP7081 originating from water in Spain (Acc. No. MK516961). The partial dnaX sequences of three Serbian P. versatile isolates (Pv1320, Pv1520, and Pv1620) and one P. carotovorum subsp. carotovorum (Pcc2520) were deposited in GenBank under Acc. No. MW839571, MW805306, MW839572, and MW805307, respectively. These results, indicating combined infection in the observed field, signify the first identification of P. versatile in Serbia. Multilocus sequence analysis (MLSA) performed with proA (proAF1/ proAR1) and mdh (mdh2/mdh4) genes (Ma et al. 2007; Moleleki et al. 2013) grouped three tested Serbian potato P. versatile isolates together with P. versatile strains from NCBI (Fig.S2). For both tested genes, BLASTn search revealed 100% homology with P. versatile strain SCC1 from Finland. Three Serbian P. versatile potato isolates were deposited under Acc. Nos. MZ682623-25 for proA and MZ682620-22 for mdh genes. According to the routine tests suggested for Pectobacteriaceae (Schaad et al. 2001), Serbian isolates possessed microbiological traits identical to P. versatile description (Portier et al. 2019). Pathogenicity was performed on potato cultivar VR808 with three selected P. versatile isolates (Pv1320, Pv1520, and Pv1620) in the following assays: (i) surface-sterilized tuber slices with holes in the center filled with 100 µL of bacterial suspensions (adjusted to 109 CFU mL-1) to test the isolates' ability to cause soft rot, and (ii) young, four-week old plants with developed 3rd true leaf (c. 30 cm tall) were inoculated by injecting stems with bacterial suspension adjusted to 107 - 108 CFU mL-1 at a height 5 cm above the soil line. Negative controls were treated with sterile distilled water. Inoculated plants were kept under controlled conditions (25 °C temperature and >70% relative humidity). Each assay was replicated twice. Soft rot appeared on tuber slices 24 h after inoculation. On inoculated stems, initial symptoms manifested as greasy elongated spots at inoculation sites two days after inoculation (DAI), and subsequently extended along the vascular tissue and became necrotic. Whole plant's decay was recorded in five DAI, while negative controls remained healthy. To complete Koch's postulates, bacteria were re-isolated from symptomatic potato plants and confirmed by PCR and sequencing of dnaX. This first report of P. versatile in potato indicates that blackleg currently present in Serbia is caused by a diverse bacterial population. This pathogen was first identified in genome comparison as 'Candidatus Pectobacterium maceratum' (Shirshikov et al. 2018) and was later renamed as Pectobacterium versatile sp. nov. (Portier et al. 2019). Thus far, bacterium Pectobacterium carotovorum subsp. brasiliensis has been recognized as dominant pathogen on most of the infected fields in Vojvodina province, and was recently noted on one plot subjected to a combined infection with Dickeya dianthicola (Marković et al. 2021). Findings achieved in this study are highly relevant, as they point to the diversity in potato blackleg pathogens, likely due to the increasingly widespread distribution of imported seed potatoes.