Pathogenicity and diversity of vegetative compatibility of Fusarium verticillioides

Pathogenicity of 10 Fusarium verticillioides isolates, originated from grain of wheat (five isolates) and maize (five isolates), were studied under greenhouse conditions. Based on different parameters of the pathogenicity estimate (a scale for % of nonemerged plants, % of survived plants, plant vigour - the growth and dry weight of roots and epicotyls and disease severity) it was determined that all F. verticillioides isolates expressed a different degree of pathogenicity. According to % of nonemerged plants six three and one F. verticillioides isolates expressed low, moderate and high degree of pathogenicity, respectively. All F. verticillioides isolates reduced the plant survival rate and vigour, while the disease severity ranged from 2.0 to 3.54. Two types of nit mutants, nit1 and NitM, were obtained by the use of the method of vegetative compatibility. The frequency of nit1 mutants was greater (58.79%) than the frequency of NitM mutants (5.77%). A total of 10 vegetative compatibility groups (VCGs) of F. verticillioides were established in the complementation tests. These results point out to a high genetic diversity of F. verticillioides population

Plesni i mikotoksini u uskladištenom kukuruzu

In this study the presence of moulds and mycotoxins in samples of stored maize grains in the period from October 2011 to September 2012 was investigated. Mycological analyses of whole and broken grains showed the presence of species from the genera Alternaria, Aspergillus, Fusarium, Penicillium, Rhizopus and others. Among the Aspergillus and Fusarium genera as potentially toxigenic fungi, Aspergillus flavus was identified with the highest percentage on broken grains (20.38%) whereas F. verticilioides was the predominant species in the whole maize grains (34.04%). In addition, it was obtained that tested samples of stored maize grains were 100% positive with aflatoxin B1 (AFB1), zearalenone (ZON), deoxynivalenol (DON) and fumonisin B1 (FB1) with an average concentration of 1.39 μg kg-1, 71.79 μg kg-1, 128.17 μg kg-1, and 1610.83 μg kg-1, respectively. A significant positive correlation was found between the moisture content and the presence of Fusarium spp. on the broken grains (r = 0.44) and between the moisture content and the concentration of DON (r = 0.61). However, a significant negative correlation was found between moisture content and FB1 (r = -0.34), and between the concentration of ZON and DON mycotoxins (r = -0.58).U radu je ispitivano prisustvo plesni i mikotoksina u uzorcima zrna uskladištenog kukuruza u periodu od oktobra 2011. do septembra 2012. godine. Mikološkim analizama celog i slomljenog zrna kukuruza ustanovljeno je prisustvo vrsta iz rodova Alternaria, Aspergillus, Fusarium, Penicillium, Rhizopus i drugih. Od potencijalno toksigenih vrsta iz rodova Aspergillus i Fusarium, identifikovane su u najvećem procentu A. flavus na slomljenom (20,38%) i F. verticillioides na celom zrnu kukuruza (34,04%). Ispitivani uzorci uskladištenog kukuruza bili su 100% pozitivni sa aflatoksinom B1 (AFB1), zearalenonom (ZON), deoksinivalenolom (DON) i fumonizinom B1 (FB1) sa prosečnim koncentracijama 1,39 μg kg-1, 71,79 μg kg-1, 128,17 μg kg-1 i 1610,83 μg kg-1, respektivno. Statistički značajna pozitivna korelacija ustanovljena je između sadržaja vlage i prisustva Fusarium spp. na slomljenom zrnu kukuruza (r = 0,44), kao i između sadržaja vlage i koncentracije DON (r = 0,61). Statistički značajna negativna korelacija ustanovljena je između sadržaja vlage i FB1 (r = -0,34), kao i između koncentracija ZON i DON mikotoksina (r = -0,58)

Fusarium infekcija i deoksinivalenol kontaminacija ozime pšenice

In this paper, the incidence of Fusarium-infected grain of winter wheat and the content of mycotoxin deoxynivalenol (DON) was studied in two Serbian cultivars Simonida and NS40S, both harvested in 2014. The level of Fusarium contamination of wheat grain was determined using phytopathological techniques based on the standard methodology while DON was detected by enzyme-linked immuno-sorbent assay (ELISA). The incidence of Fusarium-infected grain ranged from 12 to 19% for Simonida and NS40S, respectively. Fusarium graminearum, as well-known producer of DON mycotoxin, was identified among Fusarium species. In addition, Alternaria spp. was isolated in high percentage, with an average incidence of 53% (Simonida) to 63% (NS40S).The average content of DON ranged from 424 μg kg-1 to 1101 μg kg-1 for Simonida and NS40S cultivars, respectively. Statistically insignificant negative correlation (r = - 0.18) was determined between Fusariuminfected grain and DON in the cultivar Simonida and statistically insignificant positive correlation (r = 0.11) in the cultivar NS40S. The mean levels of DON in studied wheat samples of both tested cultivars were not higher than the maximum permitted limit (1250 μg kg-1) although the level of Fusarium-infected grain of both cultivars was relatively high. These results indicate that both wheat cultivars are susceptible to Fusarium infection and DON mycotoxin production in agro-ecological conditions of Serbia, but the cultivar NS40S being more susceptible compared to cultivar Simonida. In view of all stated above, regular health check of grains and developing strategies for integrated monitoring of incidence of Fusarium head blight are necessary preventive measures in protection of winter wheat.U radu je proučavana incidenca Fusarium-zaraženih zrna ozime pšenice iz žetve 2014. godine kod dve domaće komercijalne sorte Simonida i NS40S, kao i sadržaj mikotoksina deoksinivalenola (DON). Nivo fuzariozne kontaminacije zrna pšenice određen je primenom fitopatoloških testova standardne metodologije a DON je detektovan primenom imunoadsorpcione enzimske metode (ELISA). Incidenca Fusarium-zaraženih zrna bila je u proseku za sve ispitivane uzorke od 12% (Simonida) do 19% (NS40S). Od Fusarium vrsta identifikovana je jedino Fusarium graminearum, kao dobro poznati producent DON mikotoksina. Pored ove gljivične vrste, u visokom procentu izolovana je Alternaria spp. sa prosečnom incidencom od 53% (Simonida) do 63% (NS40S). U ispitivanim uzorcima pšenice prosečan sadržaj DON bio je od 424 μg kg-1 (Simonida) do 1101 μg kg-1 (NS40S). Između Fusarium-zaraženih zrna i DON utvrđena je statistički neznačajna negativna korelacija (r = - 0.18) kod sorte Simonida i statistički neznačajna pozitivna korelacija (r = 0.11) kod sorte NS40S. Prosečne koncentracije DON u ispitivanim uzorcima pšenice kod obe ispitivane sorte nisu bile iznad maksimalno dozvoljenog limita iako je nivo fuzariozne kontaminacije zrna obe ispitivane sorte bio visok. Ovi rezultati ukazuju da su obe ispitivane sorte pšenice osetljive prema fuzarioznoj infekciji i produkciji DON mikotoksina u agroekološkim uslovima Srbije, s tim što je sorta NS40S osetljivija u odnosu na sortu Simonida. Zbog svega navedenog, redovna zdravstvena kontrola zrna i razvijanje strategije integralnog monitoringa fuzarioze klasa neophodne su preventivne mere borbe u zaštiti pšenice

Morphological description and molecular detection of Pestalotiopsis sp on hazelnut in Serbia

In autumn 2015, hazelnut plants with leaf blight symptoms were noticed in a commercial plantation in the Province of Vojvodina, Serbia. Symptomatic samples were collected and submitted to laboratory analysis. Based on morphological characterization, the fungus isolated from the material was initially identified as Pestalotiopsis sp. Pathogenicity tests showed that two selected isolates infected hazelnut leaves and fruits that developed symptoms after artificial inoculation. The pathogen was re-isolated from diseased leaves and fruits, confirming Koch's postulates. Molecular identification was performed with sequence and phylogenetic analysis of ITS, EF1-alpha, and TUB genomic regions. Phylogenetic analysis confirmed the results of the morphological identification. The detection of Pestalotiopsis sp., a causal agent of leaf blight on hazelnut in Serbia, is one of a few reports of these pathogenic fungi on hazelnut

Prisustvo deoksinivalenola u ozimoj pšenici tretiranoj s fungicidima

Natural occurrence of Fusarium spp. and concentrations of mycotoxin deoxynivalenol (DON) in the grain of the winter wheat moderately susceptible to Fusarium head blight (FHB) has been studied. Grain samples were collected from wheat crops intended mainly for human consumption. All wheat crops were treated with fungicides before (a.i. flutriafol - formulated as Fluoco, applied in dose of 0.5 l ha-1) and during the flowering phase of growing (a.i. thiophanate-methyl + epoxiconazole formulated as Eskorta plus and a.i. thiophanate-methyl formulated as Funomil, applied in doses of 0.75 and 0.5 l ha-1, respectivily). Among of Fusarium species only F. graminearum, as a well known producer of DON, was identified. This fungus was identified in 15 of 19 samples (78.9%) with incidence in positive samples of 2 to 28% (average, 14.0%). Presence of DON was established in 13 of a total 19 investigated wheat grain samples (68.4%). In positive samples DON was detected in concentrations from 69 to 918 μg kg-1 (average, 478 μg kg-1). DON showed a significant and positive correlation at P≥0.05 with grain moisture content (r = 0.52*). Between the frequency of F. graminearum and concentration of DON and between the frequency of F. graminearum and grain moisture content, positive correlation was determined, but without statistical significance (r = 0.44 and r = 0.29, respectively).U radu je proučavana prirodna pojava Fusarium spp. i koncentracija mikotoksina deoksinivalenola (DON) u zrnu ozime pšenice srednje osetljive prema fuzariozi klasa (FHB). Uzorci zrna su prikupljeni sa proizvodnih useva pšenice namenjene uglavnom za ljudsku upotrebu. Svi usevi pšenice bili su tretirani sa fungicidima pre (a.m. flutriafol - formulisana kao preparat Fluoco, primenjen u dozi 0,5 l ha-1) i tokom cvetanja biljaka (a.m. tiofanat-metil + epoksikonazol formulisana kao preparat Eskorta plus i a.m. tiofanat-metil formulisana kao Funomil, primenjeni u dozi 0.75 i 0.5 l ha-1, respektivno). Među Fusarium vrstama jedino je identifikovana F. graminearum, koja je poznati producent DON. Ova gljiva je bila identifikovana u 15 od 19 uzoraka (78.9%) sa incidencom od 2 do 28% (prosek 14.0%) u pozitivnim uzorcima. Prisustvo DON je utvrđeno u 13 od ukupno 19 proučavanih uzoraka pšenice (68.4%). U pozitivnim uzorcima DON je detektovan u koncentracijama od 69 do 918 μg kg-1 (prosek 478 μg kg-1). DON je pokazao značajnu i pozitivnu korelaciju pri P≥0.05 sa sadržajem vlage zrna (r = 0.52*). Između učestalosti F. graminearum i koncentracije DON i učestalosti F. graminearum i sadržaja vlage zrna utvrđena je, takođe, pozitivna korelacija ali statistički nije značajna (r = 0.44 i r = 0.29, respektivno)

Prirodna mikobiota i prisustvo aflatoksina B1 u polenu prikupljenom u Srbiji

Total fungal count, incidence of fungi and aflatoxin B1 (AFB1) concentration were studied in 33 samples of bee pollen randomly collected from beekeepers in Serbia. The total number of fungi was determined by dilution method whereas AFB1 was detected using the Enzyme-Linked Immuno-Sorbent Assay (ELISA). The mycological estimation showed the presence of nine genera of fungi as followed: Acremonium, Alternaría, Aspergillus, Cladosporium, Epiccocum, Fusarium, Mucor, Pénicillium and Rhizopus, with total number ranging from 1 x 103 to 1 x 105 CFU g-1. The results have shown the predominance of the fungi from the genera Aspergillus and Alternaria. Among Aspergillus species it was observed that the most frequent species was A. flavus with incidence of 27.27 %. Mycotoxin AFB1 was detected as 100% positive in all samples (100%) with an average concentration of 8.61 μg kg-1. The obtained results indicated that honey bee pollen must be strictly controlled during its manipulation in the harvesting and manufacturing. Therefore, the implementation of good manufacturing (beekeeping) practice to define procedures for honeybee products could be crucial to reduce the risk of possible contamination and provide natural and safety product without risk on the human health.Ukupan broj gljiva, učestalost (incidenca) gljiva i koncentracija aflatoksina B1 (AFB1) ispitivani su u 33 uzoraka polena sakupljenih od pčelara iz različitih regiona u Srbiji. Ukupan broj gljiva određen je primenom metode razređenja a AFB1 je određen primenom imunoadsorpcione enzimske metode (ELISA). Mikološkim ispitivanjima identifikovano je devet rodova gljiva: Acremonium, Alternaria, Aspergillus, Cladosporium, Epiccocum, Fusarium, Mucor, Penicillium i Rhizopus, sa ukupnim brojem od 1 x 103 to 1 x 105 CFU g-1. Najučestalije vrste gljiva su u rodovima Aspergillus i Alternaria. Među Aspergillus vrstama najučestalija je vrsta A. flavus sa incidencom od 27,27%. AFB1 je detektovan u svim uzorcima sa prosečnom koncentracijom od 8,61 μg kg-1. Dobijeni rezultati ukazuju da pčelarski polen mora biti strogo kontrolisan tokom prikupljanja i njegove dalje prerade. Zbog toga, sprovođenje dobre proizvođačke (pčelarske) prakse podrazumeva definisanje procedura za pčelarske proizvode što bi moglo biti presudno za smanjenje rizika od moguće kontaminacije i dobijanje prirodnih i bezbednih proizvoda bez rizika po zdravlje ljudi

Toksigeni potencijal izolata Fusarium poae poreklom sa pšenice

Eleven isolates of F. poae, originated from wheat grain at 9 locations mainly in Vojvodina, were encompassed by the present study. The greatest number of samples was collected in 2005, in which the climatic conditions favoured a more intensive occurrence of Fusarium ear blight of wheat. In order to determine toxicological potential of this species, cultures of the selected isolates were grown in liquid media (GPY and SPY) on a rotary shaker (180 revolutions min-1), at room temperature (21-26°C) for three days. Crude toxins were isolated from liquid culture filtrates of isolates by the use of ethyl acetate, while quantification of mycotoxins was done by the thin layer chromatography method. A liquid culture of the isolate GZ-LES (F. graminearum) was used as a control for the evaluation of the zearalenone biosynthesis potential. On the other hand, the liquid culture of the isolate KF-38/1 (F. sporotrichioides) was used as a control for both type-A trichothecenes (T-2 toxin and diacetoxyscirpenol - DAS). The obtained results show that F. poae, in contrast to F. graminearum, has no potential for the zearalenone biosynthesis. The presence of DAS was determined only in one isolate of F. poae (MRIZP-666), and in the control isolate of F. sporotrichioides (KF-38/1/R), that were grown in the GPY liquid medium. The T-2 toxin was detected in the isolate MRIZP-666, grown in both media, and in the isolates MRIZP-37 and MRIZP-860, cultured in the GPY and SPY liquid medium, respectively. The control culture KF-38/1/R (F. sporotrichioides) produced the T-2 toxin at the concentration of 4,000 μg L-1. According to the gained information, it can be concluded that the potential of F. poae for the type-A trichothecene biosynthesis was low, as the concentration of DAS or T-2 toxin did not exceed 80 μg L-1 or 240 μg L-1, respectively.U ovom radu je u in vitro uslovima proučena sposobnost izolata F. poae za biosintezu jedne grupe fuzariotoksina - trihotecena tipa A (T-2 toksin i diacetoksiscirpenol - DAS), kao i zearalenona (ZEA). Toksikološki profil ove vrste je kod nas nedovoljno ispitan s obzirom na njenu zastupljenost i toksigena svojstva prema literaturnim podacima. Proučavanjima je bilo obuhvaćeno 11 izolata F. poae, poreklom sa pšenice iz 9 lokaliteta, uglavnom sa područja Vojvodine. Najveći broj uzoraka prikupljen je 2005. godine, kada su klimatski uslovi pogodovali intenzivnijoj pojavi fuzarioza klasa ove poljoprivredne kulture. Za određivanje toksikološkog potencijala F. poae kulture odabranih izolata su gajene u tečnim podlogama (GPK i SPK) tokom 3 dana na sobnoj temperaturi (21-26°C) i na rotacionoj tresilici (180 obrtaja min-1). Sirovi toksini su izolovani iz filtrata tečnih kultura ispitanih izolata pomoću etil acetata, dok je kvantifikacija mikotoksina izvršena metodom tankoslojne hromatografije. Tečna kultura izolata GZ-LES (F. graminearum) je korišćena kao kontrolna kultura za utvrđivanje potencijala za biosintezu zearalenona, a KF-38/1/R (F. sporotrichioides) za oba trihotecena tipa A (T-2 toksin i DAS). Dobijeni rezultati pokazuju da F. poae, za razliku od F. graminearum, ne poseduje potencijal za biosintezu zearalenona. Prisustvo DAS-a je utvrđeno samo kod jedne kulture F. poae (MRIZP-666) i kontrolnog izolata F. sporotrichioides (KF-38/1/R) koji su gajeni u tečnom GPK medijumu. T-2 toksin je detektovan kod izolata MRIZP-666 pri gajenju u obema podlogama, kao i izolata MRIZP-37 u GPK, odnosno MRIZP-860 u SPK medijumu. Kontrolna kultura KF-38/1/R (F. sporotrichioides) proizvodila je T-2 toksin u koncentraciji od 4000 μg L-1. Na osnovu iznetih podataka može se zaključiti da je potencijal F. poae za biosintezu trihotecena tipa A bio nizak u datim uslovima s obzirom da koncentracija DAS-a nije prelazila 80 μg L-1, odnosno T-2 toksina 240 μg L-1. Imajući u vidu prikazane rezultate, smatramo da je za dobijanje konačnog odgovora na pitanje o toksikološkom profilu izolata F. poae u Srbiji neophodno preduzeti dodatna ispitivanja, ne samo sa novim izolatima iz godina koje slede, nego i u drugim uslovima kultivisanja, u prvom redu na sterilnom prirodnom supstratu kao što su pšenica i kukuruz

Učestalost toksigenih vrsta Fusarium i fuzariotoksina u zrnu pšenice u Srbiji

A total of 88 and 40 wheat samples collected immediately prior to harvest in 2005 and 2006, respectively, under different agroecological conditions, were studied in respect to the occurrence of Fusarium spp. and the production of fusariotoxins. The greatest number of samples was infected with species of the genera Fusarium (81.8 and 65.0%), and Alternaria (36.3 and 17.5%) with the intensity ranging from 9.4 to 84.0% in 2005 and from 23.4 to 80.6% in 2006. Out of 13 identified species belonging to the genus Fusarium, F. graminearum had the highest frequency (35.2 and 12.5%) and the intensity up to 67.2%, and 21.9%, in 2005 and 2006, respectively, followed by F. poae but only in 2005 (20.4%), and F. proliferatum in 2006 (19.7%). The natural occurrence of mycotoxins in positive samples varied from 37 to 331 ppb for zearalenone and from 31 to 125 ppb for diacetoxyscirpenol (DAS) and T-2 toxin. The concentration of mycotoxins amounted, on average, to 133.4, 61.0 and 45.7 ppb for zearelenone, DAS and T-2 toxin, respectively.Osamdeset osam i 40 uzoraka pšenice, prikupljenih neposredno pred žetvu u 2005. i 2006. godini u različitim agroekološkim uslovima u Srbiji proučavani su radi praćenja pojave Fusarium spp. i stvaranja fuzariotoksina. Najveći broj uzoraka bio je zaražen vrstama roda Fusarium (81,8 i 65,0%), a zatim sa Alternaria spp. (36,3 i 17,5%) sa intenzitetom 9,4-84,0% u 2005. i 23,4-80,6% u 2006. godini. Od ukupno 13 identifikovanih vrsta iz roda Fusarium najučestalija je bila F. graminearum (35,2 i 12,5%) sa intenzitetom do 67,2% u 2005. i do 21,9% u 2006. godini, a zatim F. poae, ali samo u 2005. godini (20,4%), i F. proliferatum u 2006. godini (19.7%). Prirodna pojava mikotoksina u pozitivnim uzorcima je varirala od 37 do 331 ppb za zearalenon i od 31 do 125 ppb za diacetoksiscirpenol (DAS) i T-2 toksin. U proseku, koncentracija mikotoksina je bila 133,4 ppb za zearelonon, 61,0 ppb za DAS i 45,7 ppb za T-2 toksin

Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world's leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1-893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/cmice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocularmucosa was well tolerated without signs of inflammation. N-PmpC- specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFN gamma immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage

Effect of bacterial seed inoculation on nitrogen dynamics, number of bacteria in soil under maize, and maize yield

The aim of this study was to determine the effects of the inoculation of maize seeds with mixtures of bacteria (Azotobacter chroococum, Azotobacter vinelandi, Bacillus megaterium, Bacillus licheniformis) during different phenophases (6-7 leaves, silking and wax ripeness stage) and on the nitrogen dynamics, total number of microorganisms, number of azotobacter and aminoheterotrophs in Chernozem and grain yield of maize hybrid ZP 684 during 2006, 2007 and 2008. Nitrogen amount in soil was significantly higher in 2006, due to favourable meteorological conditions for microbial activity (higher total number of microorganisms and number of azotobacter), than in 2007 and 2008. The minimum amount of nitrogen was in the stage of wax ripeness although the total number of microorganisms, azotobacter and aminoheterotrophs were the largest. Seed inoculation of maize significantly increases the values of all studied parameters