Aspergillus parasiticus Speare – toksigena vrsta gljive na zrnu kukuruza u proizvodnim uslovima Srbije

Abstract

U toku četvorogodišnjih ispitivanja (2013-2016) identifikovana je, po prvi put u Srbiji, vrsta Asperigillus parasiticus kao prouzrokovač aspergilozne truleži zrna i klipa kukuruza u polju i skladištima. Sa ciljem da se ispita rasprostranjenost i diverzitet ove vrste obavljena su proučavanja morfoloških, patogenih, molekularnih, odgajivačkih i toksigenih svojstava 46 odabranih izolata ove vrste poreklom sa zrna kukuruza. Ispitivanjem makroskopskih morfoloških karakteristika izolata ukazano je na mali biodiverzitet proučavanih izolata. Kolonije izolata na PDA hranljivoj podlozi formirale su obilnu tamnozelenu, gustu prevlaku od micelije gljive, pamučastog izgleda. Svi izolati A. parasiticus obrazuju jednoćelijske, nazubljene konidije na MEA hranljivoj podlozi. Prečnik konidija ispitivanih izolata varirao je od 4,08µm do 6,86µm. Dvofaktorska analiza varijanse pokazala je statistički značajan uticaj interakcije ispitivanih izolata, svetlosnog režima i temperature (p<0.01) na dnevni porast prečnika kolonija. Test patogenosti ukazao je da su svi ispitivani izolati A. parasiticus patogeni na klipu kukuruza, kao i da postoji divergentnost u ispoljavanju virulentnosti. Poređenjem koncentracija sintetisanih mikotoksina kod izolata dobijenih Elisa testom, pokazali su da postoji statistički visoko značajna pozitivna korelacija između potencijala produkcije AFB1 i AFLA kod ispitivanih izolata (r=0,66**). Ispitivani izolati A.parasiticus koji su sintetisali aflatoksine (50%) su u najvećem procentu sintetisali AFB1 (84,78%), dok je procenat izolata koji su sintetisali AFG1 bila znatno manja (15,22%). Međutim, utvrđeno je osam izolata koji su sintetisali AFG1 u višim koncentracijama, u odnosu na AFB1. Poređenjem koncentracija mikotoksina kod izolata A. parasiticus, na osnovu rezultata HPLC metode, uočeno je da postoji visoko statistički značajna pozitivna korelacija između sintetisanih koncentracija AFB1 i AFG1 (r=0,82**), kao i između AFB2 i AFG2 (r=0,63**). Multiplex PCR detekcijom potvrđen je identitet svih izolata prethodno okarakterisanih na osnovu morfoloških, ekoloških, odgajivačkih i toksigenih karakteristika kao vrsta A. parasiticus. Primenom RFLP-PCR metode korišćenjem restrikcionih enzima, omogućeno je razlikovanje vrste A. parasiticus od morfološki slične vrste A. flavus, što je ujedno i prva identifikacija ove patogene vrste na molekularnom nivou u Srbiji.

In the course of four-year studies (20132016), the species Aspergillus parasiticus was identified in Serbia for the first time as a causal agent of aspergillus kernel and ear rot in both fields and storages. In order to examine the distribution and diversity of this species, the study of morphological, pathogenic, molecular and toxigenic properties of 48 isolates of this species selected from maize kernels was carried out. Studies of macroscopic morphological traits of isolates showed that biodiversity of observed isolates was low. Colonies of the isolates formed on PDA the abundant dark green, dense cottony mycelium coating. All isolates of A. parasiticus formed unicellular denticulate conidia on MEA. The diameter of conidia of observed isolates varied from 4.08µm to 6.86µm. Two factorial analysis of variance showed that the effects of interactions of studied isolates, light regime and temperatures (p<0.01) on daily growth of the colony diameters were statistically significant. The pathogenicity test pointed out that all observed isolates of A. parasiticus were pathogens of maize ears, as well as that there was divergence in the virulence expression. The comparison of concentrations of synthesised mycotoxins in isolates obtained by ELISA showed a statistically highly significant positive correlation between potential to produce AFB1 and AFLA in tested isolates (r=0,66**). The observed isolates A. parasiticus that synthesised aflatoxins (50%) mostly synthesised AFB1 (84.78%), while the number of isolates that synthesised AFG1 was significantly lower (15.22%). However, it was established that eight isolates synthesised AFG1 at higher concentrations than AFB1. Mycotoxin concentrations in A. parasiticus isolates were compared by using the results obtained by the HPLC method. The comparison indicated that there was a highly statistically positive correlation between synthesised concentrations of AFB1 and AFG1 (r=0.82**), as well as between AFB2 and AFG2 (r=0.63**). The identity of all isolates previously characterised on the basis of morphological, ecological and toxigenic properties as species A. parasiticus was confirmed by the multiplex PCR detection method. Distinguishing between species A. parasiticus and morphologically similar species A. flavus was enabled by the application of the RFLP-PCR method, using restriction enzymes. This was also the first identification of this pathogenic species at the molecular level in Serbia. Accepted