In Europe, two fastidious phloem-limited pathogens, ’Candidatus Phytoplasma solani’ (16SrXII-A) and ’Candidatus Arsenophonus phytopathogenicus’, are associated with rubbery taproot disease (RTD) and syndrome basses richesses (SBR) of sugar beet, respectively. Both diseases can significantly reduce yield, especially when accompanied by root rot fungi. This study investigates the presence, geographic distribution and genetic traits of fastidious pathogens and the accompanying fungus, Macrophomina phaseolina, found on sugar beet across four geographically separated plains spanning seven countries in Central Europe. The survey revealed variable incidences of symptoms linked to these fastidious pathogens in the Pannonian and Wallachian Plains, sporadic occurrence in the North European Plain, and no symptomatic sugar beet in the Bohemian Plain. Molecular analyses unveiled the occurrence of both ’Ca. P. solani’ and ’Ca. A. phytopathogenicus’ throughout Central Europe, with a predominance of the phytoplasma. These fastidious pathogens were detected in all six countries surveyed within the Pannonian and Wallachian Plains, with only a limited presence of various phytoplasmas was found in the North European Plain, while no fastidious pathogens were detected in Bohemia, aligning with observed symptoms. While 16S rDNA sequences of ’Ca. P. solani’ remained highly conserved, multi-locus characterization of two more variable loci (tuf and stamp) unveiled distinct variability patterns across the plains. Notably, the surprising lack of variability of tuf and stamp loci within Central Europe, particularly the Pannonian Plain, contrasted their high variability in Eastern and Western Europe, corresponding to epidemic and sporadic occurrence, respectively. The current study provides valuable insights into the genetic dynamics of ’Ca. P. solani’ in Central Europe, and novel findings of the presence of ’Ca. A. phytopathogenicus’ in five countries (Slovakia, Czech Republic, Austria, Serbia, and Romania) and M. phaseolina in sugar beet in Slovakia. These findings emphasize the need for further investigation of vector–pathogen(s)–plant host interactions and ecological drivers of disease outbreaks.

Penicillium crustosum Thom. is a fungus commonly found on cheese and nuts, but is also a postharvest pathogen that causes blue mold disease of pome and stone fruits including plum and nectarine (Louw and Korsten 2016; Restuccia et al. 2006). The fungus produces mycotoxins (penitrem A, roquefortine C, terrestric acid, and cyclopenol) which are of concern for human health (Frisvad and Samson 2004). In Serbia, P. crustosum has been previously described on apple fruit (Vico et al. 2014). On nectarine fruit (Prunus persica var. nucipersica), after 6 weeks of cold storage, symptoms of blue mold developed in a fruit market in Belgrade, Serbia. The fruit was collected and isolations performed in November 2017. Decayed areas on infected fruit were soft, light to medium brown with blue-green sporulation on the fruit surface. Two isolates were obtained (N2AS and N2BS) and cultured on Czapek yeast autolysate agar (CYA), malt extract agar (MEA), yeast extract sucrose agar (YES) and potato dextrose agar (PDA) at 25°C for 7 days. Isolates were identified as P. crustosum based on morphological features (Frisvad and Samson 2004; Pitt and Hocking 2009). On all media, mycelia were white and colonies turned blue-green with abundant sporulation. Colonies of both isolates were radially sulcate on MEA and YES, and plane with a granular texture on CYA and PDA, and were yellow to orange on the reverse side on YES. Mean colony diameter on PDA was 29.2 ± 1.2 mm for N2AS, and 31.3 ± 1.4 mm for N2BS; on CYA 30.8 ± 1.2 mm for N2AS and 30.9 ± 1.1 mm for N2BS; on YES 40.7 ± 3.6 mm for N2AS and 43.6 ± 1.4 mm for N2BS; and on MEA 33.4 ± 1.2 mm for N2AS and 34 ± 2.5 mm for N2BS. Crusts of conidial masses formed on MEA and PDA after 10 days. Conidiophores of both isolates were terverticillate, stipes were septate with rough walls, and conidia, borne in columns, were smooth and spherical to subglobose. Conidial diameter for N2AS was 2.32 to 3.95 (average 3.13) µm and for N2BS was 2.34 to 3.98 (average 3.27) µm (n=50). Isolates formed a yellow ring, using Ehrlich's reagent, indicating lack of cyclopiazonic acid, but production of other alkaloids. Morphological identification was confirmed by isolating genomic DNA, PCR amplification of the partial β-tubulin gene using Bt2a/Bt2b (Glass and Donaldson 1995) and sequencing. BLAST analysis revealed that N2AS sequence (MT799805) was 99% similar and N2BS (MT799806) was identical to sequences AY674351 (strain CBS 101025) and KJ775121 (strain DTO_244H8) of P. crustosum in GenBank. Sequences (2X consensus) of the two isolates differed in one nucleotide showing the existence of single-nucleotide polymorphism among P. crustosum isolates. Pathogenicity was tested on nectarine, peach and apple fruit (four fruit per isolate and the control). Fruit were washed, surface-sanitized with 70% ethanol, and wound (10x4 mm) inoculated on two sides with 40 μl of a 105/ml conidial suspension in sterile distilled water containing 0.1% Tween 20 (TSDW). Control fruit was inoculated with TSDW. Inoculated and control fruit were stored at 25°C for 7 days. Inoculated fruit developed light brown decay with cracks in the epidermis that spread from the inoculation point on nectarines and peaches. Blue-green sporulation was present on all inoculated fruit. Control fruit remained symptomless. The fungus was re-isolated and was morphologically identical to the original isolates, thus completing Koch's postulates. This is the first report of P. crustosum causing postharvest blue mold decay on nectarine fruit in Serbia. Results show that P. crustosum is not only present as a postharvest pathogen of apple fruit, but of nectarine as well and may pose a threat in storage of both pome and stone fruits in Serbia. References: Frisvad, J. C. and Samson, R. A. 2004. Stud. Mycol. 49:1. Glass, N.L. and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61: 1323. Louw, J.P., and Korsten, L. 2016. Eur. J. Plant Pathol. 146: 779. Pitt, J. I. and Hocking, A. D. 2009. Fungi and food spoilage, 239. Springer. Restuccia et al. 2006. J. Food Prot. 69: 2465. Vico, I., et al. 2014. Plant Dis. 98:1430. Acknowledgment: This research was supported by the project III46008, No. 451-03-68/2020-14/200116, financed by the Ministry of Education, Science and Technological Development, Republic of Serbia.

Blue mold, caused by Penicillium spp., is one of the most economically important postharvest diseases of pome fruits, globally. Pome fruits, in particular apple, is the most widely grown pome fruit in Serbia, and the distribution of Penicillium spp. responsible for postharvest decay is unknown. A two-year survey was conducted in 2014 and 2015, where four pome fruits (apple, pear, quince, and medlar) with blue mold symptoms were collected from 20 storage locations throughout Serbia. Detailed morphological characterization, analysis of virulence in three apple cultivars, and multilocus phylogeny revealed three main Penicillium spp. in order of abundance: P. expansum, P. crustosum, and P. solitum. Interestingly, P. expansum split into two distinct clades with strong statistical support that coincided with several morphological observations. Findings from this study are significant and showed previously undocumented diversity in blue mold fungi responsible for postharvest decay including the first finding of P. crustosum, and P. solitum as postharvest pathogens of quince and P. crustosum of medlar fruit in the world, and P. expansum of quince in Serbia. Data from this study provide timely information regarding phenotypic, morphological and genotypic plasticity in P. expansum that will impact the design of species-specific detection tools and guide the development of blue mold management strategies.

Botryosphaeria dothidea (Moug.:Fr.) Ces. & De Not. is predominantly recognized as a pathogen of various woody plants, inducing symptoms of stem canker, dieback, and fruit rot worldwide. However, sporadic reports suggest its impact on field crops, including B. dothidea associated with stem canker in soybean and tobacco (Bian et al. 2015; Chen et al. 2021), as well as B. quercuum on sugar beet (Alfieri et al. 1984). In September 2023, during a survey of root rot pathogens of sugar beet (Beta vulgaris L.) in Rimski Šančevi, Serbia (N 45°19´57″; E 19°49'58″), 3% of collected samples exhibited root rot symptoms. Externally, the lesions exhibited a dark brown coloration. On cross-section, the tissue displayed a gradient of discoloration ranging from light to dark brown throughout the roots. The roots were completely rotted. From these samples, two fungal isolates (SR28/II and SR4/III) were obtained from rotted internal root fragments, after washing, surface disinfection (70% ethanol), and plating on potato dextrose agar (PDA). Colonies on PDA were fluffy with abundant aerial mycelium, surface light to dark grey-brown, reverse black in the centre and grey-brown towards the irregular margin, after 7 days at 25°C in the dark. To induce sporulation, isolates were cultivated on 2% water agar with pine needles and incubated under continuous near ultraviolet (NUV) light at room temperature for 30 days. Conidia were hyaline, aseptate, fusiform, subtruncate at the base, subobtuse at the apex, and measured (19.56-) 24.12 - 26.31 (-28.99) x (5.13-) 5.94 - 6.54 (-7.44) µm (mean 25.06 x 6.26 µm, n=100), consistent with description of B. dothidea (Phillips et al. 2013). For molecular identification, DNA was extracted from mycelium of 7-day-old cultures, and internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha gene (TEF) and partial β-tubulin gene (TUB) were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Multiple sequence alignment and BLAST analyses showed that our isolates had 100% sequence similarity with reference isolates of B. dothidea, including ex-type isolate CBS 115476 (= CMW 8000) in ITS (AY236949), TEF (AY236898), and TUB (AY236927). Maximum likelihood phylogeny of concatenated sequences confirmed the identity of the isolates as B. dothidea. Sequences of ITS, TEF and TUB of isolate SR4/III were submitted to GenBank under accession numbers PP908658, PP911334, PP911333, respectively. The pathogenicity of obtained isolates was assessed on 3-month-old sugar beet plants grown in sterile substrate in the greenhouse. For inoculation, the upper parts of the roots were wounded (10x3 mm) 1 cm above the substrate, using a sterilized nail, and 2x2 mm mycelial plugs of 7-day-old culture grown on PDA were inserted and sealed with parafilm. Control plants were inoculated with sterile PDA plugs. Six plants were used per isolate and for control. After 3 weeks of incubation, the inoculated plants were removed from the substrate and brown rot symptoms were observed upon cross-sectional examination, whereas control roots remained asymptomatic. The pathogen was successfully reisolated and morphologically identified as B. dothidea, fulfilling Koch's postulates. The production of sugar beet, an important industrial crop in Serbia valued for its sugar content, is imperiled by various pathogens. To our knowledge, this is the first report of B. dothidea causing root rot of sugar beet in Serbia and the world. Given its widespread distribution on other hosts and potential aggressiveness, the presence of B. dothidea in sugar beet should not be neglected.

Paeonia peregrina Mill. and Paeonia tenuifolia L. are endangered species of herbaceous plants with high ornamental and medicinal values. There are a few natural habitats in Serbia where these plants spontaneously grow: Krivi Vir, Skrobnica, Golina, Pirot, Deliblato Sands, and Sokobanja, in the municipality of Knjaževac. In July 2021, the symptom of leaf spots was observed on P. peregrina, while spots on leaves, stems, and lower branches on P. tenuifolia in the localities of Pirot and Deliblato Sands. Disease incidence was estimated to 32% in Pirot and 25-35% in Deliblato Sands. Therefore, the aim of the study was to identify the causal agent of leaf spot on P. peregrina and P. tenuifolia in Serbia by molecular identification and characterization, and morphological characterization. For molecular identification and characterization, nuclear ribosomal internal transcribed spacer (ITS) region, partial translation elongation factor 1-alpha (EF-1α), beta-tubulin (TUB2) and histone 3 (H3) genes were amplified using primer pairs ITS1/ITS4, EF1-728F/EF1-986R, T1/Bt2b, and H3-1a/H3-1b, respectively. Morphological characterization of the representative isolates was done on potato dextrose agar (PDA) and potato carrot agar (PCA) media at 22 ◦C under an 8 h light:16 h dark regime. Based on the molecular and morphological characteristics of the obtained isolates, Alternaria alternata was identified as thecausal agent of leaf spot on P. peregrina and P. tenuifolia. In Serbia, to the best of our knowledge, this is the first report of A. alternata causing a leaf spot of P. tenuifolia.