Diversity of Botryosphaeriaceae and Diaporthe Species Associated with Postharvest Apple Fruit Decay in Serbia.

Family Botryosphaeriaceae and the genus Diaporthe (family Diaporthaceae) represent diverse groups of plant pathogens, which include causal agents of leaf spot, shoot blight, branch and stem cankers, dieback, and pre- and postharvest apple fruit decay. Apple fruit with symptoms of light to dark brown decay were collected during and after harvest from 2016 to 2018. Thirty selected isolates, on which pathogenicity was confirmed, were identified and characterized based on multilocus phylogeny and morphology. Five species from the family Botryosphaeriaceae and two from the genus Diaporthe (fam. Diaporthaceae) were discovered. The most commonly isolated was Diplodia seriata followed by Botryosphaeria dothidea. In this work, Diaporthe rudis is described as a new postharvest pathogen of apple fruit. Diplodia bulgarica, Diplodia sapinea, Neofusicoccum yunnanense, and Diaporthe eres are initially described as postharvest apple and D. sapinea as postharvest quince and medlar fruit pathogens in Serbia. Because species of the family Botryosphaeriaceae and the genus Diaporthe are known to cause other diseases on their hosts, have an endophytic nature, and have a wide host range, findings from this study imply that they may become a new challenge for successful fruit production.

Selection of Non-Mycotoxigenic Inulinase Producers in the Group of Black Aspergilli for Use in Food Processing.

Research background: Inulinases are used for fructooligosaccharide production and they are of interest for both scientific community and industry. Black aspergilli represent a diverse group of species that has use for enzyme production, in particular some species are known as potent inulinase producers. Finding new potential producers from the environment is as important as improving the production with known strains. Safe use of enzymes produced by aspergilli in food industry is placed ahead of their benefit for inulinase production. Experimental approach: Here we show a specific approach to finding/screening of newly isolated fungal inulinase producers that combines a newly developed screening method and an equally important assessment of the toxigenic potential of the fungus. In this study 39 black aspergilli collected from different substrates in Serbia were identified and assessed for inulinase production. Results and conclusions: The most common species were Aspergillus tubingensis (51.2%), followed by A. niger (23.1%), A. welwitschiae (23.1%) and A. uvarum (2.6%). The isolates for inulinase production were selected using a cheap and easy, fast and non-hazardous alternative inulinase screening test developed in this work. Enzymatic activity of selected inulinase-producing strains was confirmed spectrophotometrically. Since some A. niger and A. welwitschiae strains are able to produce mycotoxins ochratoxin A (OTA) and fumonisins (FB), the toxigenic potential of selected inulinase producers was assessed analytically and genetically. Fungal enzyme producer can be considered safe for use in food industry only after comparing the results of both approaches for investigating toxic potential, the direct presence of mycotoxins in the enzyme preparation (analytically) and the presence of mycotoxin gene clusters (genetically). In some strains the absence of OTA and FB production capability was molecularly confirmed by the absence of complete or critical parts of biosynthetic gene clusters, respectively. The two best inulinase producers and mycotoxin non-producers (without mycotoxin production capability as additional safety) were selected as potential candidates for further development of enzyme production. Novelty and scientific contribution: The presented innovative approach for the selection of potential fungal enzyme producer shows that only non-toxigenic fungi could be considered as useful in food industry. Although this study was done on local isolates, the approach is applicable globally.

Sensitivity of Trichoderma strains from edible mushrooms to the fungicides prochloraz and metrafenone.

Twenty-two strains of Trichoderma spp. (T. harzianum species complex [THSC], Trichoderma aggressivum f. europaeum, Trichoderma pleuroti, and Trichoderma pleuroticola) causing green mold disease on edible mushrooms (button mushroom, shiitake and oyster mushroom), collected during 2004-2018 from four countries (Serbia, North Macedonia, Croatia, and Hungary) were examined. Based on their ITS (internal transcribed spacer) sequences, strains from shiitake mushroom in Serbia were identified as members of the THSC, while in samples obtained from Serbian and North-Macedonian oyster mushroom farms THSC, T. pleuroti and T. pleuroticola were detected, which represent the first findings in the region. In fungicide susceptibility tests, all examined Trichoderma strains were found to be highly sensitive to prochloraz (ED50<0.4 µg mL-1) and considerably susceptible to metrafenone (ED50 < 4 µg mL-1). The most sensitive taxon to both fungicides was THSC from oyster mushroom. The toxicity of metrafenone was satisfying and strains from oyster mushroom showed the highest sensitivity (ED50 < 1.43 µg mL-1), while strains originating from button mushroom and shiitake displayed similar susceptibilities (ED50 < 3.64 µg mL-1). After additional in vivo trials, metrafenone might also be recommended for the control of green mold disease in mushroom farms.

First report of blue mold caused by Penicillium crustosum on nectarine fruit in Serbia.

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 ß-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.

Chamomile Floricolous Downy Mildew Caused by Peronospora radii.

Floricolous downy mildews (Peronospora, oomycetes) are a small, monophyletic group of mostly inconspicuous plant pathogens that induce symptoms exclusively on flowers. Characterization of this group of pathogens, and information about their biology, is particularly sparse. The recurrent presence of a disease causing flower malformation which, in turn, leads to high production losses of the medicinal herb Matricaria chamomilla in Serbia has enabled continuous experiments focusing on the pathogen and its biology. Peronospora radii was identified as the causal agent of the disease, and morphologically and molecularly characterized. Diseased chamomile flowers showed severe malformations of the disc and ray florets, including phyllody and secondary inflorescence formation, followed by the onset of downy mildew. Phylogeny, based on internal transcribed spacer and cox2, indicates clustering of the Serbian P. radii with other P. radii from chamomile although, in cox2 analyses, they formed a separate subcluster. Evidence pointing to systemic infection was provided through histological and molecular analyses, with related experiments validating the impact of soilborne and blossom infections. This study provides new findings in the biology of P. radii on chamomile, thus enabling the reconstruction of this floricolous Peronospora species' life cycle.

Distribution and Characterization of Monilinia spp. Causing Apple Fruit Decay in Serbia.

Brown rot, caused by Monilinia spp., is an economically important pre- and postharvest disease of pome and stone fruits worldwide. In Serbia, apple is the most widely grown pome fruit, and the distribution of economically important Monilinia spp. responsible for apple brown rot is unknown. Hence, we conducted a three year survey, from 2010 to 2012, where 349 isolates were obtained from six orchards and four storage facilities from five different apple cultivars with brown rot symptoms. Morphological characterization of the isolates, multiplex PCR, and phylogenetic analysis revealed four species: M. fructigena, M. laxa, M. fructicola, and Monilia polystroma. All species were found in the orchard and in storage, with M. fructigena predominating, followed by M. polystroma. Representative isolates were analyzed in vitro and in vivo where differences in growth rate, sporulation, and virulence on apple fruit were observed. Findings from this investigation demonstrate diversity in the species responsible for pre- and postharvest apple brown rot, which has significant implications for pathogen detection and for developing disease-specific management strategies.

Geographical variations, prevalence, and molecular dynamics of fastidious phloem-limited pathogens infecting sugar beet across Central Europe.

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.

A biotroph sets the stage for a necrotroph to play: 'Candidatus Phytoplasma solani' infection of sugar beet facilitated Macrophomina phaseolina root rot.

'Candidatus Phytoplasma solani' (stolbur phytoplasma) is associated with rubbery taproot disease (RTD) of sugar beet (Beta vulgaris L.), while Macrophomina phaseolina is considered the most important root rot pathogen of this plant in Serbia. The high prevalence of M. phaseolina root rot reported on sugar beet in Serbia, unmatched elsewhere in the world, coupled with the notorious tendency of RTD-affected sugar beet to rot, has prompted research into the relationship between the two diseases. This study investigates the correlation between the occurrence of sugar beet RTD and the presence of root rot fungal pathogens in a semi-field 'Ca. P. solani' transmission experiment with the cixiid vector Reptalus quinquecostatus (Dufour), in addition to naturally infected sugar beet in the open field. Our results showed that: (i) Reptalus quinquecostatus transmitted 'Ca. P. solani' to sugar beet which induced typical RTD root symptoms; (ii) Macrophomina phaseolina root rot was exclusively present in 'Ca. P. solani'-infected sugar beet in both the semi-field experiment and naturally infected sugar beet; and that (iii) even under environmental conditions favorable to the pathogen, M. phaseolina did not infect sugar beet, unless the plants had been previously infected with phytoplasma.

Incidence, Speciation, and Morpho-Genetic Diversity of Penicillium spp. Causing Blue Mold of Stored Pome Fruits in 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.