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

Uticaj etarskih ulja timijana, cimeta i karanfilića na porast micelije Colletotrichum acutatum

Effects of the volatile phase of thyme, cinnamon and clove essential oils on Colletotrichum acutatum were investigated. Mycelial disc was placed in the center of the Petri dish (V=66 ml) containing PDA. Different volumes of either non- or ethanol-diluted essential oils were placed on the inner side of the dish cover to obtain final concentrations of 153, 107, 76, 46, 15, 14, 12, 11, 7.6, 3.82, 1.53, 0.153 and 0.0153 μl/L of air. The dishes were sealed with Parafilm and incubated in up-side-down position. After 7 days of incubation, mycelial growth was recorded by measuring the colony diameter. If no mycelial growth was recorded, the disc was transferred to a new PDA plate in order to evaluate whether the activity was either fungistatic or fungicidal. Mean growth values were obtained and then converted to inhibition percentage of mycelial growth compared with the control treatment. All the tested essential oils inhibited mycelial growth of C. acutatum in the dose dependent manner. Mycelial growth was totally inhibited by thyme oil in the concentration of 76 μl/L of air. The same results were obtained by cinnamon and clove oil in the concentration of 107 μl/L of air. Thyme and cinnamon oil had fungicidal effect in concentrations of 107 and 153 μl/L respectively. The results obtained provide evidence on the antifungal in vitro effect of the tested essential oils as potential means for the control of C. acutatum.Proučavan je efekat isparljive faze etarskih ulja timijana, cimeta i karanfilića na prouzrokovača antraknoze jagode Colletotrichum acutatum u uslovima in vitro. Fragmenti micelije gljive, prečnika 1 cm, zasejani su na KDA podlogu u petri-kutije (V = 66 ml). Različite koncentracije etarskih ulja (153, 107, 76, 46, 15, 14, 12, 11, 7,6, 3,82, 1,53, 0,153 i 0,0153 μl/l vazduha) dobijene su nanošenjem određene količine ulja, koncentrovanih ili razblaženih u etanolu, na središnji deo unutrašnje strane poklopca. Petri-kutije su zatvorene parafilmom i postavljene u obrnuti položaj, a porast micelije je meren posle 7 dana inkubacije. U kulturama u kojima nije došlo do porasta micelije fungicidno ili fungitoksično dejstvo određeno je prebacivanjem fragmenata na KDA podloge bez prisustva ulja. Efekat etarskih ulja predstavljen je procentom inhibicije porasta micelije, poređenjem sa kontrolom. Proučavana etarska ulja timijana, cimeta i karanfilića su svojom gasovitom fazom inhibirala porast micelije C. acutatum. Stepen inhibicije zavisio je od primenjene koncentracije. Etarsko ulje timijana je potpuno inhibiralo porast micelije pri koncentraciji 76 μl/l vazduha, a cimeta i karanfilića pri koncentraciji 107 μl/l vazduha. Fungicidno dejstvo ispoljilo je etarsko ulje timijana u koncentraciji 107 μl/l vazduha i cimeta u koncentraciji 153 μl/l vazduha. Rezultati istraživanja in vitro pokazali su antifungalno dejstvo testiranih etarskih ulja, što govori o potencijalu korišćenja etarskih ulja u kontroli C. acutatum

Penicillium polonicum - prouzrokovač propadanja lukovica crnog luka u skladištu

Penicillium polonicum is an economically important fungal species with a wide host range which includes cereals, peanuts, onions, dried meats, citrus, apple, and yam tubers. Onion bulbs cv. Meranto with blue mold symptoms have been collected, in a storage facility in Stara Pazova during 2013. Fungal isolates were obtained from decayed bulbs and their pathogenicity was proven after wound inoculation of healthy onion bulbs. On differential media the isolates formed velutinous, green blue colonies with a white margin and a characteristic odor. The isolates grew at 5 C while no growth was observed at 37 C. Isolates produced cyclopiazonic acid and other alkaloids. Conidiophores of isolates were terverticillate, stipes were septate with smooth to finely roughened walls, and phialides were ampulliform. Conidia were globose to subglobose, smooth-walled, and borne in columns. The nucleotide sequences of amplified products (~800 bp) for partial β-tubulin gene showed in MegaBLAST search analysis a 99% similarity with several sequences of P. polonicum. Based on morphological and molecular features isolates obtained from stored onion were identified as P. polonicum.Penicillium polonicum je ekonomski važna gljiva sa širokim krugom domaćina, uključujući žitarice, kikiriki, lukove, sušeno meso, citruse, jabuke i krtole jama. U januaru 2013. godine, u skladištu na teritoriji Stare Pazove prikupljeni su uzorci lukovica crnog luka cv. Meranto sa karakterističnim simptomima plave truleži. Iz obolelih lukovica dobijeni su izolati gljive čija je patogenost potvrđena inokulacijom zdravih lukovica. Na diferencijalnim podlogama izolati su formirali dobro razvijene, guste kolonije, plavozelene boje sa belim obodom, karakterističnog mirisa. Izolati nisu rasli na 37oC, dok je porast zabeležen na 5oC. Utvrđeno je da su izolati stvarali ciklopiazonsku kiselinu i druge alkaloide. Konidiofore izolata su bile terverticilate, stipe septirane, tankih i glatkih zidova, a fijalide cilindrične. Konidije su bile loptaste do elipsoidne, glatke i formirale su se u dugim lancima. Sekvence dobijenih amplikona veličine oko 800 bp dela gena za β-tubulin su u MegaBLAST analizi pokazali najvišu sličnost od 99% sa sekvencama istog regiona vrste P. polonicum. Na osnovu morfoloških i molekularnih osobina kao prouzrokovač propadanja lukovica crnog luka u skladištu identifikovana je gljiva P. polonicum

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

Antifungalna aktivnost etarskih ulja cimeta i karanfilića prema patogenima šampinjona - Cladobotryum dendroides (Bull.) W. Gams & Hooz i Lecanicillium fungicola var. fungicola (Preuss) Hasebrauk

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

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

Prisustvo i rasprostranjenost virusa duvana u Srbiji

Infection with a large number of plant viruses could imperil tobacco yield and quality. Tobacco is a natural host for more than 20 viruses, among which the most important and economically harmful are tobacco mosaic virus (TMV), tomato spotted wilt virus (TSWV), cucumber mosaic virus (CMV), potato virus Y (PVY), alfalfa mosaic virus (AMV), tobacco ring-spot virus (TRSV), tobacco each virus (TEV) and tobacco vein mottling virus (TVMV). The occurrence and distribution of tobacco viruses were investigated for 4 years (20022005). During this period many different tobacco growing localities in Vojvodina and central Serbia were monitored and samples showing virus symptoms were collected. The collected samples were tested by DAS ELISA using polyclonal antisera, specific for the detection of PVY, TSWV, TMV, CMV, AMV and TRSV. The results obtained for the tobacco virus distribution during these four years of investigation proved the presence of four economically important viruses in our country, whose frequencies varied from year to year. In 2002, 2003 and 2004, the most frequent was TSWV (86.84%; 79% and 49.56%, respectively), while in 2005 PVY was prevalent (56.16%). All viruses detected in the samples tested were present in single or mixed infections. A correlation was established between the field symptoms on tobacco and the virus causal agents. The results obtained showed that TSWV and PVY were the most important tobacco viruses in our country, so that further research of tobacco virus diseases should be directed towards their more detailed characterization.Prinos i kvalitet duvana mogu da budu ugroženi usled zaraze velikim brojem fitopatogenih virusa. Duvan je prirodni domaćin preko 20 virusa od kojih su najznačajniji i ekonomski najštetniji virus mozaika duvana (Tobacco mosaic virus, TMV), virus bronzavosti paradajza (Tomato spotted wilt virus, TSWV), virus mozaika krastavca (Cucumber mosaic virus, CMV), virus crtičastog mozaika krompira (Potato virus Y, PVY), virus mozaika lucerke (Alfalfa mosaic virus, AMV), virus prstenaste pegavosti duvana (Tobacco ringspot virus, TRSV), virus graviranosti duvana (Tobacco each virus, TEV) i virus šarenila nerava duvana (Tobacco vein mottling virus, TVMV). Prisustvo i rasprostranjenost virusnih zaraza duvana su ispitivani tokom četiri godine, od 2002. do 2005. Pregled i sakupljanje uzoraka sa simptomima obavljeno je u više različitih lokaliteta gajenja duvana u Vojvodini i centralnoj Srbiji. Sakupljeni uzorci su testirani DASELISA metodom na prisustvo PVY, TSWV, TMV, CMV, AMV i TRSV. Dobijeni rezultati ispitivanja ukazuju na prisustvo četiri ekonomski značajna virusa duvana u našoj zemlji i to: TSWV, PVY, TMV i CMV, čija je učestalost po pojedinim godinama i lokalitetima bila različita. Tokom 2002, 2003. i 2004. godine najčešće detektovan virus bio je TSWV (86,84%; 70%, odnosno 49,56%), dok je 2005. godine prevalentan virus bio PVY (56,16%). Svi virusi čije je prisustvo utvrđeno u ispitivanim uzorcima nalazili su se u pojedinačnim ili mešanim infekcijama. Bez obzira na izraženu varijabilnost u ispoljenim simptomima virusnih zaraza na duvanu, ustanovljena je zavisnost tipa ispoljenih simptoma od virusa prouzrokovača. Sprovedena ispitivanja pokazala su da su TSWV i PVY najznačajniji virusi duvana u našoj zemlji, tako da buduća ispitivanja viroza duvana treba da budu usmerena ka detaljnijoj karakterizaciji ovih ekonomski štetnih virusa

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

Pogodnost različitih prajmera za specifičnu molekularnu detekciju Monilinia spp.

Monilinia spp. are economically important pathogens of pome and stone fruits. Four Monilinia species are present in Serbia - Monilinia fructigena, M. laxa, M. fructicola and Monilia polystroma. As detection and identification of Monilinia species are complex, the aim of this research was to evaluate species-specific primers in PCR in order to standardize fast and reliable molecular methods for differentiation between the four Monilinia species. Isolates of M. fructigena, M. laxa, M. fructicola and M. polystroma from apple fruit and referent isolates from Italy and Japan were used for testing. Specific molecular detection of M. laxa was obtained using ITS1Mlx/ITS4Mlx and Ml-Mfg-F2/Ml-Mfc-R1 primer pairs, and M. fructicola using ITS1Mfcl/ITS4Mfcl and Mfc-F1/Mfc-R1 primer pairs. ITS1Mfgn/ITS4Mfgn and ITS1/Mfg-R2 primer pairs, described as M. fructigena species-specific, amplified M. fructigena and M. polystroma, as well. Specific detection of these two species as well as of all four tested Monilinia species was obtained using the reverse primer MO368-5 with forward primers MO368-8R, Laxa-R2 and MO368-10R in separate or in Multiplex PCR reactions.Vrste roda Monilinia su ekonomski znač ajni patogeni jabučastih i koštičavih vrsta voćaka. U Srbiji su prisutne četiri vrste ovog roda: M. fructigena, M. laxa, M. fructicola i Monilia polystroma. Detekcija i identifikacija vrsta roda Monilinia je složena, tako da je cilj ovog rada bio da se ispita i standardizuje brza i efikasna metoda molekularne detekcije korišćenjem različitih prajmera u PCR metodi i da se utvrdi njihova specifičnost i mogućnost korišćenja u identifikaciji Monilinia spp. U radu su korišćeni izolati M. fructigena, M. laxa, M. fructicola i M. polystroma poreklom iz plodova jabuke prikupljenih u Srbiji, kao i referentni izolati iz Italije i Japana. Specifična molekularna detekcija M. laxa postignuta je korišćenjem prajmera ITS1Mlx/ITS4Mlx i Ml-Mfg -F2/Ml-Mfc-R1, a vrste M. fructicola korišćenjem prajmera ITS1Mfcl/ITS4Mfcl i Mfc-F1/Mfc-R1. Prajmeri ITS1Mfgn/ITS4Mfgn i ITS1/Mfg -R2, koji su u literaturi opisani kao specifični za M. fructigena, pored izolata M. fructigena amplifikovali su i DNK izolata M. polystroma. Za razdvajanje ove dve vrste, kao i za razlikovanje sve četiri ispitivane vrste roda Monilinia, najpogodniji su uzvodni prajmer MO368-5 u kombinaciji sa nizvodnim prajmerima MO368-8R, Laxa-R2 i MO368-10R u direktnoj ili Multiplex PCR metodi