Karakterizacija antimikrobnih supstanci prirodnih izolata Bacillus sp. za primenu u biološkoj kontroli fitopatogenih bakterija i gljiva

Abstract

Bolesti biljaka izazvane infekcijama patogenim bakterijama i gljivama mogu dovesti do smanjenja kapaciteta biljnog rasta ili naneti mnogo ozbiljniju štetu dovodeći do smrti biljaka i značajnih gubitaka u proizvodnji hrane. Veliki broj istraživanja je u poslednje vreme posvećen proučavanju roda Bacillus kao producenta sekundarnih metabolita u kontroli različitih biljnih patogena. Analizom diverziteta gena odgovornih za produkciju neribozomalno sintetisanih lipopeptida kolekcije od 205 Bacillus sp. izolata utvrđeno je da je za najveći broj izolata (171) ustanovljen DNK fragment očekivane dužine, koji odgovara bacilomicin D sintetazi, dok su u sličnom broju izolata dobijeni fragmenti očekivanih dužina za kurstakinske (154) i surfaktinske operone (142), a prisustvo iturinskog operona ustanovljeno je kod 84, odnosno 59 izolata u slučaju gena za fengicin sintetazu. Prisustvo svih pet biosintetičkih operona je potvrđeno kod 11 izolata. Najveća produkcija antimikrobnih jedinjenja za pet izabranih izolata je pokazana u eksponencijalnoj ili ranoj stacionarnoj fazi rasta. Prikazan je jak antifungalni efekat lipopeptidnih ekstrakata prema više od 30 testiranih gljiva, sa najnižim zabeleženim minimalnim inhibitornim koncentracijama od 0,008 mg/ml protiv Fusarium semitectum, kao i izražen antibakterijski potencijal, naročito protiv fitopatogenih vrsta rodova Xanthomonas i Pseudomonas, sa najnižom minimalnom inhibitornom koncentracijom od 0,06 mg/ml. Proučavanje interakcija pojedinačnih lipopeptidnih ekstrakata u međusobnim, kao i u kombinacijama sa etarskim uljima, ukazalo je na postojanje sinergističkog efekta za neke kombinacije u in vitro, odnosno aditivnog efekta u in situ uslovima, na bakterijskim i fungalnim patogenima. Utvrđivanjem direktnog antagonističkog efekta lipopeptidnih jedinjenja in planta, ustanovljena je značajna redukcija nekroze tkiva listova Arabidopsis thaliana L. i Beta vulgaris L., tretiranih bakterijskim patogenima. Takođe, u in situ uslovima postignuta je značajna redukcija nekroze tkiva ploda jabuke izazvana fungalnom infekcijom...

Plant diseases caused by infection with pathogenic bacteria and fungi can lead to the reduction in the capacity of plant growth or can cause far more serious damage, leading to the death of plants and significant losses in food production. Numerous recent studies are devoted to the genus Bacillus as a producer of secondary metabolites in the control of different plant pathogens. In the analysis of diversity of genes responsible for the nonribosomal synthesized lipopeptides in collections of 205 Bacillus sp. isolates, for the largest number of isolates (171) the DNA PCR fragment of the expected length, corresponding to bacillomycin D synthases was obtained, while in a similar number of isolates fragments of the expected length for kurstakin (154) and surfactin operons (142) were obtained. The presence of iturin operon in the 84 or 59 isolates in the case of the gene for fengycin synthetase, was also found. The presence of all five biosynthetic operons in the 11 isolates was confirmed as well. The maximum production of antimicrobial compounds for five selected isolates in exponential or early stationary phase of growth was determined. Strong antifungal effect of lipopeptide extracts towards more than 30 fungi tested, with lowest recorded minimal inhibitory concentrations of 0,008 mg/ml against Fusarium semitectum, as well as expressed antibacterial potential, in particular against phytopathogenic species of the genera Pseudomonas and Xanthomonas, with the lowest minimum inhibitory concentration of 0,06 mg/ml was determined. In the analysis of the interaction of individual lipopeptide extracts mutually, as well as in combination with essential oils, the existence of a synergistic effect in vitro respectively additive effect in situ, on bacterial and fungal pathogens, for some combinations were shown. Establishing a direct antagonistic effect of lipopeptide compounds in planta, significant reduction in necrotic lesions of leaves of Arabidopsis thaliana L. and Beta vulgaris L. treated with bacterial pathogens was obtained. Also, a significant reduction in necrotic lesions of apple fruit caused by fungal infection in situ was determined...