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In Europe, the cultivation of sugar beet faces significant challenges due to the emergence of basses richesses syndrome (SBR) and rubbery taproot disease (RTD) which are associated with fastidious bacteria Candidatus Arsenophonus phytopathogenicus and Candidatus Phytoplasma solani, respectively. A comprehensive study conducted from 2018 to 2022 aimed to elucidate the prevalence and distribution of these pathogens in sugar beet in central Europe, focusing on the Pannonian Plain (particularly Serbia and Slovakia where RTD has been present in epidemic scales) and Germany.

Waitea circinata var. zeae, a pathogen with a relatively narrow host range, has recently been detected in cabbage and oilseed rape in Europe and worldwide. In this study, we developed specific conventional and real-time PCR protocols for direct detection of W. circinata var. zeae from mycelium and diseased plant tissue. The newly developed primer pair zeaefor1/zeaerew1, used in PCR protocols, specifically amplified only target isolates of W. circinata var. zeae when tested against isolates of 11 different binucleate and multinucleate anastomosis groups of Rhizoctonia spp. including AG-A, AG-G, AG-F, AG-U, AG-2-1, AG-2-2, AG-3, AG-4 HGI, AG-4 HGII, AG-4 HGIII, and AG-6 and common soil-borne pathogens. Total of nine previously published primer pairs designed for the detection of various Rhizoctonia spp. were also tested and did not amplify target isolates of W. circinata var. zeae. The detection limit of conventional and real-time PCR protocols was 10–2 and 10–5 (with starting concentration 9.5 ng/µl), respectively, and both methods are the first available tools for direct detection and identification of W. circinata var. zeae from mycelium and diseased oilseed rape seedlings. Both conventional and SYBR-Green-based real-time PCR protocols are cost-effective and provide a solid basis for further investigations of W. circinata var. zeae, particularly in relation to distribution, host range, and epidemiology.

Waitea circinata var. zeae (anamorf Rhizoctonia zeae) je značajan patogen uglavnom onokotiledonih biljaka, koji je prisutan u mnogim delovima sveta, a održava se u zemljištu duži niz godina. Poslednjih godina krug domaćina W. circinata var. zeae širi se i na neke dikotiledone biljke. U okviru nedavnih istraživanja u Srbiji W. circinata var. zeae je utvrđen kao prouzrokovač truleži korena i korenovog vrata, i propadanja biljaka kupusa i uljane repice na lokalitetima Futog i Rimski Šančevi. U radu su sistematizovani publikovani podaci o rasprostranjenosti, krugu domaćina, načinu održavanja i širenja, kao i suzbijanju W. circinata var. zeae, kao i rezultati istraživanja koja su obavljena u Srbiji.

‘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. Copyright © 2023 Duduk, Vico, Kosovac, Stepanović, Ćurčić, Vučković, Rekanović and Duduk.

U periodu 2013-2020. godine, pregledom 22 useva na 15 lokaliteta i šest partija krompira u Srbiji, ustanovljena je pojava nekroze i truleži korena različitih biljaka. Sakupljeno je 330 uzoraka i dobijeno 177 izolata Rhizoctonia spp., koji su morfološki i molekularno razdvojeni u 11 anastomoznih grupa (AG). Rezultati su potvrđeni sekvenciranjem ITS regiona rDNA, kao i LSU, rpb2, tef-1α, atp6 i β-tubulin genskih markera. Detaljno su okarakterisana 64 izolata i generisano je 77 sekvenci koje su omogućile prvi uvid u diverzitet i strukturu populacije Rhizoctonia spp. u Srbiji. Kao prouzrokovači bolesti u Srbiji javljaju se dvojedarne Rhizoctonia spp. AG-A na jagodi, AG-G na paradajzu, trešnji i pasulju, AG-F na paprici, AG-U na jabuci i Poa sp., a od višejedarnih R. solani potvrđeno je prisustvo AG-2-1 na paprici, AG-2-2 na šećernoj repi, AG-3 na krompiru, AG-4 HGI na mrkvi, paprici, uljanoj repici i kupusu, AG-4 HGII na jabuci, AG-6 na malini, kao i Waitea circinata var. zeae na kupusu i uljanoj repici. Od toga, osam AG dokazano je u Srbiji prvi put ili prvi put na novom domaćinu u Srbiji, a osam AG je prvi put opisano na novim domaćinima u svetu. Filogenetske analize ukazale su na njihove međuodnose, na moguće puteve introdukcije i postojanje varijabilnosti u okviru roda Rhizoctonia u Srbiji. Razvijen je protokol za direktnu detekciju AG primenom specifičnih prajmera, kao i qPCR protokol za specifičnu detekciju R. solani AG-2-2. Ustanovljena je različita virulentnost i krug domaćina pojedinih AG inokulacijama 14 biljaka iz osam familija. Identifikovan je izolat Trichoderma harzianum sa agresivnošću prema različitim AG u in vitro i in vivo eksperimentima. Dobijeni rezultati pokazuju da su vrste roda Rhizoctonia veoma značajna i raznovrsna grupa patogena u Srbiji.

During the 2014-2017 period, wheat samples were collected from discoloured spikes affected by Fusarium head blight (FHB) from 20 locations in Serbia. After isolation, fungi were cultivated on potato dextrose agar (PDA) at 25oC for 7 days. Based on the in situ identification on PDA, 36 isolates of the section Liseola were selected for further analyses. The pathogenicity of all isolates was confirmed on wheat leaves. The virulence of isolates was determined by measuring the lengths of spots formed at the inoculation leaf site. In order to prove the presence of the species Fusarium verticillioides, a pair of primers FV-F2/FV-R was used. This pair of primers amplifies the sequence of the gaoB gene, and it proved to be specific for the stated species. Moreover, for the same purpose, a pair of primers VER1-VER2 based on the calmodulin partial gene was used. The reference isolate RBG 1603 Q27 was used as a positive control. The pair of primers VER1-VER2 produced a band of the expected size - 578 bp in 18 isolates, while using FV-F2/FV-R, a 370bp long band confirmed the presence of F. verticillioides in 16 samples. Sixteen out of 18 isolates verified with VER1-VER2 were also identified as F. verticillioides with FV-FS/FV-R. No amplification was observed in a negative control., Tokom perioda od 2014. do 2017. godine sakupljani su uzorci pšenice sa fuzarioznih klasova sa 20 lokacija u Srbiji. Nakon izolacije, gljive su gajene na KDA podlozi 7 dana u termostatu na temperaturi od 25oC. Na osnovu identifikacije in situ na KDA (krompir dekstrozni agar), 36 izolata sekcije Liseola odabrano je za dalje analize. Potvrđena je patogenost svih izolata na listovima pšenice. Virulentnost izolata utvrđena je merenjem dužina formirane pege na mestu inokulacije. Za dokazivanje prisustva vrste Fusarium verticillioides korišćen je par prajmera FV-F2/FV-R koji umnožava sekvence gaoB gena i koji se pokazao kao specifičan za navedenu vrstu. Takođe, sa istim ciljem korišćen je par prajmera VER1-VER2 dizajniran na osnovu dela genske sekvence za kalmodulin. Kao pozitivna kontrola korišćen je referentni izolat RBG 1603 Q27. Par prajmera VER1-VER2 obrazovao je traku očekivane veličine (578bp) kod 18 izolata, dok je pomoću para prajmera FV-F2/FV-R traka dužine 370bp potvrdila prisustvo F. verticillioides kod 16 izolata. Šesnaest od 18 izolata koji su identifikovani VER1-VER2 parom, takođe su identifikovani i FV-FS/FV-R prajmerima. U negativnoj kontroli nije bilo amplifikacije.

Kupus i uljana repica su dva vrlo značajna useva u našoj zemlji i njihova proizvodnja ugrožena je brojnim patogenim organizmima. Višejedarne anamorfne Rhizoctonia spp. sa teleomorfom opisanim kao Waitea circinata su biljni patogeni koji se dugo održavaju u zemljištu i rasprostranjeni su i vrlo štetni širom sveta. Od pet opisanih varijeteta W. circinata var. zeae je najrasprostranjeniji. Za sada poznat krug domaćina W. circinata var. zeae obuhvata monokotiledone i mali broj dikotiledonih biljaka, među kojima soju, pasulj, grašak, šećernu repu i mrkvu.

Cabbage, a widely used and popular vegetable, and oilseed rape, the second most valuable oilseed crop in the world, are two important species from the Brassicaceae family. Two geographically separated outbreaks of cabbage and oilseed rape root rot with estimated incidence of 15 and 20%, respectively, were recorded during 2017 in the Vojvodina region, Serbia. Twelve hyphal-tip isolates were obtained from symptomatic cabbage and oilseed rape plants and identified as Waitea circinata var. zeae based on morphological and molecular features. This indicates that W. circinata var. zeae has expanded its host range to the Brassicaceae family. Sequence analyses of internal transcribed spacer (ITS) and large subunit of the ribosomal DNA, RPB2, and B-tubulin genes revealed the highest similarity with multiple W. circinata var. zeae. Neighbor-joining analyses of ITS sequences resulted in a phylogenetic tree with one welldefined branch of W. circinata var. zeae, with two separate groups. All Serbian isolates and the majority of isolates originating from natural infection of dicotyledonous plants grouped together in group I. Following artificial inoculation, W. circinata var. zeae isolates caused mild to medium root necrosis of seedlings of 2 monocotyledonous and 12 dicotyledonous plant species, implying a wider host range than was known for W. circinata var. zeae. Additionally, this is the first occurrence of W. circinata var. zeae on dicotyledonous host plants in Europe. Because cabbage and oilseed rape are important crops grown worldwide, the occurrence of this new soilborne pathogen with a broad host range imposes the necessity for changes in routine disease control practices, particularly crop rotation.

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.

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 (ED(50)0.4 mu g mL(-1)) and considerably susceptible to metrafenone (ED50 lt 4 mu g 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 lt 1.43 mu g mL(-1)), while strains originating from button mushroom and shiitake displayed similar susceptibilities (ED50 lt 3.64 mu g mL(-1)). After additional in vivo trials, metrafenone might also be recommended for the control of green mold disease in mushroom farms.

Penicillium expansum-infected apple fruit resulted in a disrupted balance of primary metabolites and impacted the ascorbate-glutathione cycle. The initial decline in fructose, malic acid, shikimic acid and total ascorbate content (6 hpi) was followed by a significant increase in malic acid (24 hpi) and total glutathione (3 dpi). Significant increase in sucrose was accompanied by a decline in glucose and ascorbate content (5 dpi). Total glutathione and shikimic acid gradually deteriorated with symptom development. A negative correlation was found between sucrose and ascorbate-glutathione pool. Other correlations were strong amongst other metabolites and lesion size indicating that they may be involved or important for apple fruit decay.

Soil-borne fungi belonging to the genus Rhizoctonia are considered to be among the most destructive sugar beet pathogens. Although multinucleate R. solani AG-2-2 is frequently detected as the main causal agent of root rot of sugar beet worldwide, several binucleate (AG-A, AG-E and AG-K) and multinucleate Rhizoctonia (R. solani AG-4, AG-5 and AG-8) have also been included in the disease complex. Due to their soil-borne nature and wide host range, the management of Rhizoctonia root rot of sugar beet is highly demanding. Identification of Rhizoctonia AG associated with root rot of sugar beet is the essential first step in determining a successful disease management strategy. In this paper we report a highly specific and sensitive real-time PCR protocol for detection of R. solani AG-2-2 which showed a high level of specificity after testing against 10 different anastomosis groups and subgroups, including AG-2-1 as the most closely related. Moreover, a similar conventional PCR assay showed the same specificity but proved to be at least a 100 times less sensitive. Future research will include further testing and adaptation of this protocol for direct detection and quantification of R. solani AG-2-2 in different substrates, including plant tissue and soil samples., Smatra se da su gljive iz roda Rhizoctonia koje se održavaju u zemljištu, najdestruktivniji patogeni šećerne repe. Mada je širom sveta višejedarna R. solani AG-2-2 najčešće označena kao najvažniji prouzrokovač truleži korena šećerne repe, nekoliko dvojedarnih (AG-A, AG-E i AG-K) kao i višejedarnih Rhizoctonia (R. solani AG-4, AG-5 i AG-8) takođe mogu da učestvuju u kompleksu bolesti. Kako se održavaju u zemljištu i imaju širok krug domaćina, suzbijanje vrsta Rhizoctonia koje izazivaju trulež korena šećerne repe veoma je zahtevno. Identifikacija anastomoznih grupa prouzrokovača oboljenja predstavlja neophodan prvi korak u uspostavljanju uspešnog suzbijanja. U ovom radu razvijen je visokospecifičan i osetljiv real-time PCR protokol za detekciju i identifikaciju R. solani AG-2-2 koji se pokazao kao visokospecifičan nakon testiranja koja su obuhvatila 10 različitih AG grupa i podgrupa, uključujući i AG-2-1 kao najsrodniju. Sličan konvencionalni PCR protokol ispoljio je istu specifičnost, ali i najmanje 100 × manju osetljivost. Buduća istraživanja uključiće dalje testiranje i adaptaciju ovog protokola za direktnu detekciju i kvantifikaciju R. solani AG-2-2 u različitim uzorcima, uključujući biljno tkivo i zemljište.

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.

Bull's eye rot caused by Neofabraea spp. is an important postharvest disease of apple fruit worldwide. Four species of the genus are known to cause this disease among which Neofabraea alba is the main pathogen causing bull's eye rot in continental Europe. Typical symptoms of the disease were observed in a local market in Belgrade, Serbia in March 2015. Circular lesions, slightly sunken light brown to dark brown with a lighter brown to tan center and a darker outer ring were present on 'Golden Delicious' apples. Decayed tissue was firm. The aim of this study was to identify the causal agent of bull's eye rot of apple fruit based on morphological and molecular characteristics of the pathogen. Two fungal isolates were obtained using standard laboratory procedure and their pathogenicity was tested by wound inoculation of healthy 'Idared' apple fruit. Seven days post inoculation lesions up to 23 cm in diameter developed on inoculated fruit while control fruit remained healthy. The isolates were preliminary identified based on colony morphology. They formed round, slow growing colonies on potato dextrose agar (PDA) with initially white mycelium which turned pinkish-brown with time. Cylindrical to curved-fusiform macroconidia were formed. Microconidia were not observed. Species level identification was completed by sequence analysis of the partial beta-tubulin gene. MegaBLAST analysis of the obtained nucleotide sequences (622 nt) revealed 100% and 99% similarity with several Neofabraea alba sequences deposited in GenBank. Based on morphological characteristics and the partial sequence analysis of p-tubulin gene, Neofabraea alba (Guthrie) Verkley was identified as the causal agent of bull's eye rot of apple fruit 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.

Strawberry production is a popular, fast-growing agricultural business in Serbia. Its cultivar selection has been changing fast, following market demands. One of the limiting factors of strawberry production worldwide is black root rot, primarily caused by binucleate Rhizoctonia. Recently, outbreaks of black root rot of strawberry have occurred in Serbia and the estimated disease incidence was up to 30%. Isolates of binucleate Rhizoctonia AG-A were recovered from symptomatic strawberry plants, and characterized on the bases of morphological, molecular and pathogenic features. Despite their uniform morphological characteristics, the isolates demonstrated genetic variability within ITS rDNA, grouping into three different phylogenetic sub-clusters which comprise AG-A isolates originating from Italy, Israel, Japan and the USA. The binucleate Rhizoctonia AG-A from Serbia exhibited uniform virulence on strawberry after inoculation of daughter plants and detached leaf petioles, as well as on seedlings of bean, carrot and sunflower, while they were non-pathogenic to wheat, maize, tomato, pepper, tobacco, cucumber, lettuce, peas, cabbage, rapeseed and sugar beet., Proizvodnja jagode je popularna grana poljoprivrede u Srbiji koja se brzo razvija. Sastav sorti uključenih u proizvodnju podleže stalnim promenama u skladu sa zahtevima tržišta. Jedan od ograničavajućih faktora u proizvodnji jagode širom sveta je crna trulež korena, bolest koju najčešće izazivaju dvojedarne vrste roda Rhizoctonia. U Srbiji je poslednjih godina nekoliko puta zabeležena pojava crne truleži jagode uz učestalost koja je iznosila do 30%. Iz biljaka jagode sa simptomima izdvojeni su izolati dvojedarne Rhizoctonia AG-A i okarakterisani na osnovu morfološih i molekularnih osobina i patogenosti. Uprkos uniformnim morfološkim osobinama, izolati su ispoljili genetičku varijabilnost na osnovu sekvence ITS rDNK, grupišući se u tri različita filogentska sub-klastera koji su obuhvatali isolate iz Italije, Izraela, Japana i SAD. Izolati dvojedarnih Rhizoctonia AG-A poreklom iz Srbije ispoljili su uniformnu virulentnost na jagodi nakon inokulacija živića i fragmenata lisnih peteljki, kao i na sejancima pasulja, mrkve i suncokreta, dok nisu bili patogeni za pšenicu, kukuruz, paradajz, papriku, duvan, krastavac, salatu, grašak, kupus i šećernu repu.

Antifungal activity of two essential oils, cinnamon (Cinnamomum verum J. Presl) and clove (Syzygium aromaticum (L.) Merrill & Perry), was evaluated against Cladobotryum dendroides (Bull.) W. Gams & Hooz, and Lecanicillium fungicola var. fungicola (Preuss) Hasebrauk, the causal agents of cobweb and dry bubble disease of cultivated mushroom. Inhibitory and fungicidal activity of the selected essential oils was assayed using three methods: microdilution, macrodilution fumigant and macrodilution contact method. Comparing all three methods, clove essential oil showed stronger activity than cinnamon against both fungi, having minimum inhibitory concentration (MIC) at the lowest concentrations tested (1.56, 0.02 and 0.1 µl ml-1, respectively). However, cinnamon oil was more toxic to L. fungicola var. fungicola then to C. dendroides in all three methods. Both oils exhibited stronger antifungal effects when used in the macrodilution fumigant than in contact method. The results showed that both cinnamon and clove essential oils have the potential for further in vivo experiments against L. fungicola var. fungicola and C. dendroides and indicated a possible use of these oils in integrated disease management in mushrooms., Ispitana je antifungalna aktivnost etarskih ulja cimeta i karanfilića prema Cladobotryum dendroides (Bull.) W. Gams & Hooz, i Lecanicillium fungicola var. fungicola (Preuss) Hasebrauk, prouzrokovačima paučinaste plesni i suve truleži šampinjona. Antifungalna aktivnost odabranih etarskih ulja testirana je primenom tri različite metode: mikrodilucione, makrodilucione fumigantne i makrodilucione kontaktne metode. Etarsko ulje karanfilića je ispoljilo jači efekat od ulja cimeta na obe gljive primenom sve tri metode, sa minimalnim inhibitornim koncentrecijama pri najnižim testiranim koncentracijama (1,56, 0,02 i 0,1 µl ml-1). Ulje cimeta je pokazalo veću toksičnost prema L. fungicola var. fungicola nego prema C. dendroides. Ispitivana etarska ulja ispoljila su jače antifungalno dejstvo primenjena fumigantno nego kontaktno u makrodilucionoj metodi. Antifungalno delovanje etarskih ulja cimeta i karanfilića in vitro, ukazuje na potencijal za dalja ispitivanja njihove efikasnosti in vivo, kao i mogućnosti uključivanja ovih etarskih ulja u integralnu zaštitu jestivih gljiva od bolesti prouzrokovanih mikopatogenim gljivama L. fungicola var. fungicola i C. dendroides.