Utvrđivanje prisustva Colletotrichum acutatum u latentno zaraženom lišću i lisnim drškama jagode

Colletotrichum acutatum is the most significant agent of anthracnose strawberry fruit rot. Besides being a necrotrophic pest, it can spend a part of its life cycle as an epiphyte, in a form of latent infection. The presence of the fungi on symptomless plant tissue is considered one of the main ways of distribution of this economically harmful pathogen in the world. Investigation of latent C. acutatum infection was carried out on artificially inoculated strawberries. The initiation of fungi sporulation on symptomless petioles and leaves was carried out by exposing them to the herbicide paraquat (0.25%) and low temperatures, which caused plant tissue decay in different ways. Surface sterilization with 0.5% NaOCl precedes the exposure of plant material to paraquat. The freezing procedure was carried out by exposure of plant material to the temperature of -20°C for 2h. After the freezing, one group was rinsed in Tween 20 (18 μl/l), and another group underwent surface sterilization in 0.0525% NaOCl with an addition of Tween 20 (18 μl/l). After 6 days of incubation, the appearance of acervuli and conidia was detected in 93.33 to 100% plant parts exposed to paraquat treatment and freezing procedure. In inoculated parts which were not exposed to herbicides or low temperatures, the presence of acervuli was detected in 3.33% tested petioles and 6.67% leaves.Colletotrichum acutatum je najznačajniji prouzrokovač antraknoznog propadanja plodova jagode. Pored nekrotrofnog načina parazitiranja, deo svog životnog ciklusa može da provede kao epifit i to u vidu latentnih zaraza. Prisustvo gljive na asimptomatičnom biljnom tkivu smatra se jednim od glavnih načina širenja ovog ekonomski štetnog patogena u svetu. Ispitivanja latentne zaraze C. acutatum urađena su na veštački inokulisanim jagodama. Iniciranje sporulacije gljive na asimptomatičnim lisnim drškama i lišću urađeno je izlaganjem biljnog tkiva delovanju herbicida parakvata, kao i niskim temperaturama, koji na različite načine izazivaju propadanje biljnog tkiva. Izlaganju asimptomatičnih biljnih delova delovanju parakvata (0,25%), prethodila je površinska sterilizacija 0,5% NaOCl. Postupak smrzavanja urađen je izlaganjem biljnih delova temperaturi od -20oC u trajanju od 2 h. Posle smrzavanja jedna grupa je isprana Tween 20 (18 μl/l), a druga grupa je površinski sterilisana 0,0525% NaOCl uz dodatak Tween 20 (18 μl/l). Posle šest dana inkubacije, pojava acervula i konidija detektovana je u 93,33% do 100% lisnih delova izloženih delovanju parakvata ili postupcima smrzavanja. U inokulisanim biljnim delovima koji nisu izlagani delovanju herbicida ili niskih temperatura, sporulacija je detektovana kod 3,33% testiranih lisnih drški, odnosno 6,67% lišća

Morfološke, serološke i molekularne analize prouzrokovača antraknoze ploda banane

Two species of the genus Colletotrichum, C. musae and C. gloeosporoides, occur as infecting species of banana. The study focused on examining the etiology of anthracnose on banana fruits sold on the domestic market. An isolate was obtained from a deseased banana fruit on PDA medium, forming a white colony with intensive and uniformed growth. It was not possible to identify the isolated fungus based on its morphological characteristics. Positive serological reaction in an ELISA test with monoclonal antibodies for C. acutatum indicated an antigen site for the used monoclonal antibodies. Positive reaction when C. gloeosporioides-specific primers were applied indicated a similarity in the ITS sequence of the fungus and the examined isolate from banana fruit. Although there are no available data in literature that C. gloeosporioides-specific CgInt primer can be used for amplification of the phylogenetically related C. musae, our results do not exclude that the isolate could be C. musae. The host plant, symptoms observed and colony characteristics of the fungus isolated from the banana fruit mostly correspond to C. musae. Based on morphological, antigen and gentic characteristics, the isolate from banana was determined as Colletotrichum sp., while species identification of the anthracnose-causing agent on banana requires additional analysis.Na banani se javljaju dve vrste iz roda Colletotrichum, C. musae i C. gloeosporoides. Cilj ovog rada bio je ispitivanje etiologije antraknoznog propadanja plodova banane koje se u našoj zemlji nalaze u prometu. Iz obolelog ploda banane dobijen je izolat koji na PDA podlozi formira koloniju bele boje sa intenzivnim uniformnim rastom. Na osnovu dobijenih morfoloških osobina izolovane gljive nije se mogla definitivno uraditi identifikacija. Pozitivna serološka reakcija u ELISA testu sa monoklonalnim antitelima za C. acutatum govori o postojanju antigenog mesta za korišćena monoklonalna antitela. Pozitivna reakcija korišćenjem prajmera specifičnih za C. gloeosporioides ukazuje na sličnost u sekvenci ITS regiona ove gljive i ispitivanog izolata iz banane. Mada u literaturi nije zabeleženo da se korišćenjem CgInt prajmera specifičnog za C. gloeosporioides može amplifikovati i njemu filogenetski bliska C. musae, ovi rezultati ne isključuju mogućnost da je izolat iz banane C. musae. Biljka domaćin, zabeleženi simptomi i osobine kolonije izolata iz banane najviše odgovaraju C. musae. Na osnovu dobijenih rezultata morfoloških, antigenih i genetskih osobina izolat iz banane označen je kao Colletotrichum sp., a za tačnu identifikaciju prouzrokovača antraknoze ploda banane do nivoa vrste neophodno je sprovesti dalje analize

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

Antraknoza - nova bolest jagode u Srbiji i njena kontrola fungicidima

Anthracnose is a destructive disease of strawberry fruits in warm and continental climate. During 2004, in the vicinity of Valjevo, there were severe losses in two strawberry plantations due to fruit anthracnose. Two fungal isolates, GG-6A and GG-JUP were recovered from strawberry stolons and fruits showing severe anthracnose symptoms. Based on morphological and pathological characteristics, and PCR analyses with specific primers of reference species, isolate GG-6A was identified as Colletotrichum gloeosporioides, and GG-JUP isolate as C. acutatum. This is the first identification of C. acutatum in strawberry in Serbia. In order to control strawberry anthracnose, five fungicides and their combinations were applied four times during the flowering. The best fruit protection was achieved by fungicides Metiram + piraclostrobin (Cabrio top), Captan FL and Fludioksinil + ciprodinil (Swich). Less effective were Benomil (Benlate) and Krezoksim-metil (Stroby). Pathogen is transmitted by planting material, so phytosanitary measures are extremely important in preventing the disease.Antraknoza je destruktivna bolest plodova jagode u toplim i kontinentalnim klimatskim uslovima. Tokom 2004. godine, na 2 plantaže jagoda u blizini Valjeva, bilo je velikih gubitaka prouzrokovanih antraknozom. Dva izolata gljiva GG-6A i GG-JUP su izolovana iz stolona jagode, i plodova sa izraženim simptomima antraknoze. Na osnovu morfoloških i patoloških karakteristika, i PCR analize sa specifičnim prajmerima za referentne vrste, izolat GG-6A je identifikovan kao Colletotrichum gloeosporioides, a izolat GG-JUP kao C. acutatum. Ovo je prvi nalaz C. acutatum na jagodi u Srbiji. U cilju kontrole antraknoze jagode pet fungicida i njihovih kombinacija su primenjeni 4 puta tokom cvetanja. Najbolja zaštita plodova jagode je postignuta primenom fungicida Metiram + piraclostrobin (Cabrio top), Captan FL i Fludioksinil ciprodinil (Swich). Manje efektivni su bili Benomil (Benlate) i Krezoksim-metil (Stroby). Patogen se prenosi sadnim materijalom pa su fitosanitarne mere veoma važne u prevenciji bolesti

Current View on Phytoplasma Genomes and Encoded Metabolism

Phytoplasmas are specialised bacteria that are obligate parasites of plant phloem tissue and insects. These bacteria have resisted all attempts of cell-free cultivation. Genome research is of particular importance to analyse the genetic endowment of such bacteria. Here we review the gene content of the four completely sequenced ‘Candidatus Phytoplasma' genomes that include those of ‘Ca. P. asteris' strains OY-M and AY-WB, ‘Ca. P. australiense,' and ‘Ca. P. mali'. These genomes are characterized by chromosome condensation resulting in sizes below 900 kb and a G + C content of less than 28%. Evolutionary adaption of the phytoplasmas to nutrient-rich environments resulted in losses of genetic modules and increased host dependency highlighted by the transport systems and limited metabolic repertoire. On the other hand, duplication and integration events enlarged the chromosomes and contribute to genome instability. Present differences in the content of membrane and secreted proteins reflect the host adaptation in the phytoplasma strains. General differences are obvious between different phylogenetic subgroups. ‘Ca. P. mali' is separated from the other strains by its deviating chromosome organization, the genetic repertoire for recombination and excision repair of nucleotides or the loss of the complete energy-yielding part of the glycolysis. Apart from these differences, comparative analysis exemplified that all four phytoplasmas are likely to encode an alternative pathway to generate pyruvate and ATP

Survey and multigene characterization of stolbur phytoplasmas on various plant species in Serbia

Stolbur phytoplasmas were detected in 116 out of more than 200 samples from nine plant species col- lected in 2009‒2010 in Serbia. Phytoplasmas were detected and identified by restriction fragment length polymor- phism (RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rDNA. While all strains were identi- cal on the level of 16S rDNA sequences, one strain represented a unique variant of stolbur phytoplasma with a SNP producing a new Tru1I restriction site. RFLP analyses of tuf gene amplicons, using HpaII restriction enzyme, showed profiles identical to tuf type II profile in all stolbur phytoplasma strains from Serbia. Seminested PCR for amplification of the rp gene yielded aspecific amplicons with nearly half of the samples examined. However, in the 23 samples which yielded amplicons of expected size, RFLP analysis with AluI restriction enzyme showed profiles with some variations. RFLP analyses of the amplified complete secY gene, using Tru1I and AluI restriction enzymes, showed profiles indistinguishable from each other. In phylogenetic analyses of secY gene, strains belong- ing to tuf type I formed a lineage separate from the strains belonging to tuf type II. To our knowledge this is the first report of stolbur phytoplasma in valerian (Valeriana officinalis) adding a new plant species to the already wide natural host range of stolbur phytoplasma

Anthracnose: A new strawberry disease in Serbia and its control by fungicides

Anthracnose is a destructive disease of strawberry fruits in warm and continental climate. During 2004, in the vicinity of Valjevo, there were severe losses in two strawberry plantations due to fruit anthracnose. Two fungal isolates, GG-6A and GG-JUP were recovered from strawberry stolons and fruits showing severe anthracnose symptoms. Based on morphological and pathological characteristics, and PCR analyses with specific primers of reference species, isolate GG-6A was identified as Colletotrichum gloeosporioides, and GG-JUP isolate as C. acutatum. This is the first identification of C. acutatum in strawberry in Serbia. In order to control strawberry anthracnose, five fungicides and their combinations were applied four times during the flowering. The best fruit protection was achieved by fungicides Metiram + piraclostrobin (Cabrio top), Captan FL and Fludioksinil + ciprodinil (Swich). Less effective were Benomil (Benlate) and Krezoksim-metil (Stroby). Pathogen is transmitted by planting material, so phytosanitary measures are extremely important in preventing the disease

Phytoplasmas infecting sugar beet in central Europe

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

Gumoza šećerne repe ozbiljna pretnja proizvodnji šećerne repe u centralnoj Evropi

Gumoza šećerne repe prvi put je u Srbiji primećena u srednjem Banatu i severnoj Bačkoj 1960-ih (Marić i sar. 1970). Bolest je u to vreme već bila prisutna u susednim zemljama, Bugarskoj i Rumuniji, ali je povezivana sa abiotičkim faktorima. Nakon epidemije tokom kasnih 1960-ih, bolest je ostala prisutna tokom 1970-ih, kada je sporadično primećena u celom regionu, pokazujući veću prevalenciju u sušnim sezonama. Tokom 2018. godine bolest je ušla u novu epidemijsku fazu u Srbiji, s to sugeriše da je možda povezana sa trenutnim epidemijama u drugim evropskim regionima gajenja šećerne repe. Stanje poznato u usevima šećerne repe kao bolest sa niskim sadržajem šećera (SBR), u Francuskoj se više puta javljala od 1990-ih, dok je u Nemačkoj i Švajcarskoj ova bolest primećena 2009. i 2017. godine. Procene nemačkih stručnjaka su da je 2020. godine više od 20.000 hektara zahvaćeno ovom bolešću. SBR je povezan sa prokariotskom bakterijom, Candidatus Arsenophonus phytopathogenicus, čiji je vektor Pentastiridius leporinus (Auchenorrhyncha: Cixiidae). Međutim, još jedna prokariotska bakterija, Candidatus Phytoplasma solani (stolbur fitoplazma) sporadično je bila prisutna u biljkama pogođenim SBR-om

Epidemiological role of novel and already known 'Ca. P. solani' cixiid vectors in rubbery taproot disease of sugar beet in Serbia

Rubbery taproot disease (RTD) of sugar beet was recently associated with the plant pathogenic bacterium ‘Candidatus Phytoplasma solani’ (CaPsol) and reported throughout the Pannonian Plain with variations in severity. Tracing CaPsol epidemiological pathways was performed in the experimental sugar beet field in Rimski Šančevi (Serbia) in 2020–2021, where an RTD outbreak was recently recorded. A molecular epidemiology approach was applied to the study of three RTD occurrence scenarios: epidemic, non-epidemic and ‘absence of RTD’. As a result, Hyalesthes obsoletus ex Convolvulus arvensis was detected as a CaPsol vector to sugar beet, while two other cixiids were identified for the first time as vectors of the CaPsol-induced plant disease in crops: Reptalus quinquecostatus and R. cuspidatus. R. quinquecostatus was proposed culpable for the 2020 RTD epidemic outbreak in Rimski Šančevi when dSTOLg CaPsol strain predominated in the RTD-affected sugar beet, whereas R. cuspidatus had a negligible role in RTD occurrence and displayed ambiguous involvement in CaPsol epidemiology on a wider scale. The temporal discrepancy of the offset of CaPsol dissemination and disease occurrence is the main obstacle in predicting CaPsol-induced diseases. Predicting disease occurrence and severity can only be achieved by gaining a better understanding of CaPsol epidemiological pathways and insect vectors involved in disease outbreaks.The Supplementary Material is available at [https://doi.org/10.1038/s41598-023-28562-8]