Occurrence and Identification of Pectobacterium carotovorum subsp. brasiliensis and Dickeya dianthicola Causing Blackleg in Some Potato Fields in Serbia.

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 (Backa 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.

First report of Pectobacterium versatile causing blackleg of potato in Serbia.

Potato blackleg is frequently observed on the production fields in the Backa 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 Maglic 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 (Markovic 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.

Diversity of Bacterial Soft Rot-Causing Pectobacterium Species Affecting Cabbage in Serbia.

The aim of this work was to identify and characterize the pectolytic bacteria responsible for the emergence of bacterial soft rot on two summer cabbage hybrids (Cheers F1 and Hippo F1) grown in the Futog locality (Backa, Vojvodina), known for the five-century-long tradition of cabbage cultivation in Serbia. Symptoms manifesting as soft lesions on outer head leaves were observed during August 2021, while the inner tissues were macerated, featuring cream to black discoloration. As the affected tissue decomposed, it exuded a specific odor. Disease incidence ranged from 15% to 25%. A total of 67 isolates producing pits on crystal violet pectate (CVP) medium were characterized for their phenotypic and genotypic features. The pathogenicity was confirmed on cabbage heads. Findings yielded by the repetitive element palindromic-polymerase chain reaction (rep-PCR) technique confirmed interspecies diversity between cabbage isolates, as well as intraspecies genetic diversity within the P. carotovorum group of isolates. Based on multilocus sequence typing (MLST) using genes dnaX, mdh, icdA, and proA, five representative isolates were identified as Pectobacterium carotovorum (Cheers F1 and Hippo F1), while two were identified as Pectobacterium versatile (Hippo F1) and Pectobacterium odoriferum (Hippo F1), respectively, indicating the presence of diverse Pectobacterium species even in combined infection in the same field. Among the obtained isolates, P. carotovorum was the most prevalent species (62.69%), while P. versatile and P. odoriferum were less represented (contributing by 19.40% and 17.91%, respectively). Multilocus sequence analysis (MLSA) performed with concatenated sequences of four housekeeping genes (proA, dnaX, icdA, and mdh) and constructed a neighbor-joining phylogenetic tree enabled insight into the phylogenetic position of the Serbian cabbage Pectobacterium isolates. Bacterium P. odoriferum was found to be the most virulent species for cabbage, followed by P. versatile, while all three species had comparable virulence with respect to potato. The results obtained in this work provide a better understanding of the spreading routes and abundance of different Pectobacterium spp. in Serbia.

Phylogeographic Analysis of Soft-Rot-Causing Pectobacterium spp. Strains Obtained from Cabbage in Serbia.

The aim of this study was to establish a link between genetic diversity and the geographic origin of Pectobacterium strains belonging to three species-P. carotovorum, P. versatile, and P. odoriferum-isolated from cabbage in Serbia by comparing their sequences with those of strains sourced from different hosts and countries in Europe, Asia, and North America. Phylogeographic relatedness was reconstructed using the Templeton, Crandall, and Sing's (TCS) haplotype network based on concatenated sequences of the housekeeping genes dnaX, icdA, mdh, and proA, while pairwise genetic distances were computed by applying the p-distance model. The obtained TCS haplotype networks indicated the existence of high intra-species genetic diversity among strains of all three species, as reflected in the 0.2-2.3%, 0.2-2.5%, and 0.1-1.7% genetic distance ranges obtained for P. carotovorum, P. versatile, and P. odoriferum, respectively. Five new haplotypes (denoted as HPc1-HPc5) were detected among cabbage strains of P. carotovorum, while one new haplotype was identified for both P. versatile (HPv1) and P. odoriferum (HPo1). None of the TCS haplotype networks provided evidence of significant correlation between geographic origin and the determined haplotypes, i.e., the infection origin. However, as haplotype network results are affected by the availability of sequencing data in public databases for the used genes and the number of analyzed strains, these findings may also be influenced by small sample size.

Study of the anticancer potential of Cd complexes of selenazoyl-hydrazones and their sulfur isosters.

The biological activity of Cd compounds has been investigated scarce since Cd has been recognized as a human carcinogen. However, the toxicity of cadmium is comparable to the toxicity of noble metals such as Pt and Pd. The paradigm of metal toxicity has been challenged suggesting that metal toxicity is not a constant property, yet it depends on many factors like the presence of appropriate ligands. Studies on anticancer activity of cadmium complexes showed that the complexation of various ligands resulted in complexes that showed better activities than approved drugs. In the present study, cadmium complexes with biologically potent thiazolyl/selenazoyl-hydrazone ligands have been prepared, and tested for their activity against different types of tumor cell models. The complexation of ligands with Cd(II) resulted in a synergistic effect. The antiproliferative activity study revealed that all complexes are more active compared to 5-fluorouracil and cisplatin. The mechanism of tumor cell growth inhibition reveal that selenium-based compounds induce cell death in T-47D (gland carcinoma) cells through apoptosis via caspase-3/7 activation. Additionally, their pro-apoptotic effect was stronger compared to etoposide and cisplatin. Nuclease activity, detected by gel electrophoresis, may be the possible mechanism of anticancer action of investigated complexes.

A novel binuclear hydrazone-based Cd(II) complex is a strong pro-apoptotic inducer with significant activity against 2D and 3D pancreatic cancer stem cells.

A novel binuclear Cd complex (1) with hydrazone-based ligand was prepared and characterized by spectroscopy and single crystal X-ray diffraction techniques. Complex 1 reveals a strong pro-apoptotic activity in both human, mammary adenocarcinoma cells (MCF-7) and pancreatic AsPC-1 cancer stem cells (CSCs). While apoptosis undergoes mostly caspase-independent, 1 stimulates the activation of intrinsic pathway with noteworthy down regulation of caspase-8 activity in respect to non-treated controls. Distribution of cells over mitotic division indicates that 1 caused DNA damage in both cell lines, which is confirmed in DNA interaction studies. Compared to 1, cisplatin (CDDP) does not achieve cell death in 2D cultured AsPC-1 cells, while induces different pattern of cell cycle changes and caspase activation in 2D cultured MCF-7 cells, implying that these two compounds do not share similar mechanism of action. Additionally, 1 acts as a powerful inducer of mitochondrial superoxide production with dissipated trans-membrane potential in the majority of the treated cells already after 6 h of incubation. On 3D tumors, 1 displays a superior activity against CSC model, and at 100 µM induces disintegration of spheroids within 2 days of incubation. Fluorescence spectroscopy, along with molecular docking show that compound 1 binds to the minor groove of DNA. Compound 1 binds to the human serum albumin (HSA) showing that the HSA can effectively transport and store 1 in the human body. Thus, our current study strongly supports further investigations on antitumor activity of 1 as a drug candidate for the treatment of highly resistant pancreatic cancer.

Measuring Patients' Knowledge About Adverse Effects of Angiotensin-Converting Enzyme Inhibitors.

BACKGROUND: Knowledge about adverse effects of medications is an important part of proper medication use and prerequisite for good treatment adherence. OBJECTIVE: The aim of our study was to construct, develop, and test a new questionnaire for the measurement of patients' knowledge about adverse drug reactions of angiotensin-converting enzyme (ACE) inhibitors. METHODS: The 8-item questionnaire was constructed to measure adverse reactions to ACE inhibitors. The questions were closed, with 7 offered answers, in the form of a Likert scale. It was tested for psychometric properties on patients who visited their general practitioners at state-owned health facilities in 5 Serbian cities: Belgrade, Kragujevac, Banja Luka, Gracanica, and Despotovac. RESULTS: The questionnaire was tested on 259 patients from general practice, taking an ACE inhibitor for more than 3 months. Experience with at least 1 adverse effect of ACE inhibitor was reported in 64 patients (24.7%), only 94 patients (36.3%) previously received any form of information about at least 1 adverse effect of ACE inhibitors from health workers, and only 42% expressed knowledge of any adverse events. The patients who were informed knew about the following adverse events as phrased in the official patient information leaflets: severe dizziness or light-headedness (44%); cough (37%); swelling of the hands, face, lips, or tongue (32%); indigestion (22%); headache (51%); and difficulty in breathing (15%). The questionnaire showed satisfactory internal consistency, with Cronbach α of 0.767, and individual scores correlated with general education of the patients. Factorial analysis revealed 2 domains (subscales): the first one with 5 questions is directed to adverse effects the patients may physically experience directly, whereas the second with 3 questions measures knowledge about adverse effects that could be experienced only indirectly, through conditions caused by the adverse effects. CONCLUSIONS: The questionnaire about knowledge of ACE inhibitors' adverse effects is a reliable and probably valid instrument for measuring patients' knowledge about adverse effects of ACE inhibitors.