Biološko suzbijanje Colletotrichum spp. parazitauskladištenih plodova jabuke

Abstract

Colletotrichum spp. su značajni prouzrokovači bolesti biljaka u svetu i u našoj zemlji, u polju i u skladištu. Na plodovima jabuke prisutne su dve vrste ovog roda, C. acutatum i C. gloeosporioides. Pomenute vrste uzrokuju ekonomski značajne gubitke posle berbe plodova jabuke, tokom skladištenja, transporta i plasmana na tržište. Nedoumice sa kojima se naučna javnost suočava kada je u pitanju ovaj rod jeste precizno utvrđivanje sistematske pozicije i definisanje vrsta i nižih kategorija. Klasične fitopatološke metode ne omogućavaju preciznu iv identifikaciju do nivoa vrste. Otuda su molekularni pristupi sve zastupljeniji u identifikaciji Colletotrichum spp. U suzbijanju skladišnih patogena pretežno se primenjuju hemijski fungicidi. Zbog nepovoljnih toksikoloških svojstava i pojave rezistentnosti, primena hemijskih fungicida se sve više redukuje, a njihova upotreba posle berbe plodova zabranjena je u većini zemalja. Stoga, za održivi razvoj poljoprivredne proizvodnje neophodna su istraživanja usmerena ka otkrivanju mikroorganizama i prirodno sintetisanih materija koje imaju potencijala za primenu u biološkoj zaštiti. Cilj ovog rada je da se utvrdi zastupljenost Colletotrichum spp. na uskladištenim plodovima jabuke u R. Srbiji, kao i pouzdanost klasičnih i molekularnih metoda za njihovu identifikaciju do nivoa vrste i nižih kategorija. Utvrđivanje filogenetske pozicije i genetičke udaljenosti izolata je takođe svrstano u ciljeve istraživanja. Nadalje, cilj je i da se izdvoje mikroorganizami i etarska ulja koja ispoljavaju antifungalno delovanje na Colletotrichum spp. Zatim, da se za mikroorganizam sa najizraženijim antifungalnim delovanjem definišu uslovi kultivacije (sastav hranljive podloge i trajanje) koji maksimizuju njegovu antifungalnu aktivnost i utvrdi način njegovog delovanja. Utvrđeno je da su Colletotrichum spp. redovno prisutni na uskladištenim plodovima jabuke u Vojvodini i delovima zapadne, centralne i jugoistočne Srbije i da je njihova zastupljenost u odnosu na druge fitopatogene gljive 7,8-10%. Zbog osetljivosti C. gloeosporioides na niske temperature, C. acutatum postaje sve dominantnija vrsta ovog roda na uskladištenim plodovima jabuke. Razlikovanje C. acutatum i C. gloeosporioides je moguće na osnovu fenotipskih karakteristika kolonija (u slučaju hromogenih izolata C. acutatum), dužine konidija, rasta kolonija na 5 i 35°C i brzine rasta kolonija na različitim podlogama. Oblik i širina konidija i optimalna temperatura rasta su nedovoljno pouzdani kriterijumi za identifikaciju do nivoa vrste. PCR metodom uz primenu prajmera specifičnih za vrstu uspešno se identifikuju C. acutatum i C. gloeosporioides. Primenom univerzalnih prajmera ITS1 i ITS4 amplifikuju se rDNK-ITS sekvence ovih izolata. Analizom sekvenci izrađuju se filogenetska stabla visoke stabilnosti i jasno razdvajaju C. acutatum i C. gloeosporioides, a u okviru C. acutatum se odvajaju nehromogeni i hromogeni izolati. S. hygroscopicus, S. aureus, B. cereus, P. aeruginosa i B. subtilis sojevi N146, ST 1/III, Č13 i QST 713 ispoljavaju antifungalnu aktivnost na Colletotrichum spp. in vitro i in vivo. S. hygroscopicus obezbeđuje zaštitu plodova na nivou sa hemijskim fungicidima (trifloksistrobin, boskalid+piraklostrobin, pirimetanil+flukvinkonazol, kaptan). Kultivacijom S. hygroscopicus u podlozi sa 15,07 g/l glicerola, 5,28 g/l ekstrakta kvasca i 0,81 g/l fosfata, u v trajanju 3-4 dana, postiže se maksimalno antifungalno delovanje na Colletotrichum spp. Antifungalno delovanje S. hygroscopicus zasnovano je na produkciji ekstracelularnih, termostabilnih metabolita. Dvomesečno skladištenje trtiranih plodova na 2±0,5°C ne slabi antifungalno delovanje S. hygroscopicus. Etarska ulja origana i timijana ispoljavaju snažno inhibitorno delovanje na Colletotrichum spp.

Colletotrichum spp. are significant plant pathogens worldwide in field, as well as on stored fruits. Two species of this genera, C. acutatum and C. gloeosporioides, can occur on apple fruits. The species cause significant economic losses on apple fruits after harvest, during storage, transport and marketing. Scientific community faces confusion in defining precise systematic position of this genera, as well as in identification to the species level and lower categories. Conventional viii phytopathological methods do not provide precise identification to the species level. Thus, molecular approaches are taking the lead in Colletotrichum spp. identification. Post-harvest pathogens are mainly managed by chemical fungicides. Due to adverse toxicological properties and resistance occurrence, use of chemical fungicides is being reduced, and their application after harvest is prohibited in most countries. Therefore, studies regarding detection of microorganisms and naturally synthetized substances with a potential for application in biological control are necessary for sustainable development of agriculture. The aim of this study was to determine frequency of Colletotrichum spp. on stored apple fruits in the Republic of Serbia as well as reliability of conventional and molecular methods in their identification to the species level and lower categories. Determination of phylogenetic position and molecular distance of the isolates were also set as investigation goals. The aim was also to determine microorganisms and essential oils with antifungal activity against Colletotrichum spp. Defining of cultivation conditions (nutrient medium composition and duration) for the microorganism with the most pronounced antifungal activity which maximize its activity and defining its mode of action were also set as goals. It was found that Colletotrichum spp. are commonly present on stored apple fruits in Vojvodina Province and in western, central and southeastern parts of Serbia with a share of 7.8-10% among other phytopathogenic fungi. Due to susceptibility of C. gloeosporioides to low temperatures, C. acutatum is becoming dominant species of this genus on stored apple fruits. Discrimination between C. acutatum and C. gloeosporioides is possible on the basis of phenotypic characteristics of colony (in the case of chromogenus isolates of C. acutatum), conidium length, colony growth at 5 and 35°C and colony growth rate on different media. Conidium shape and width and optimal growth temperature are insufficient criteria for identification to the species level. PCR method using species-specific primers is reliable for identification of C. acutatum and C. gloeosporioides. rDNK-ITS sequences of the isolates can be successfully amplified with universal primers, ITS1 and ITS4. Analyses of the sequences alow construction of highly stable phylogenetic trees with distinctively separated C. acutatum and C. gloeosporioides clades, and also additional clades of non-chromogenum and chromogenum C. acutatum isolates. S. hygroscopicus, S. aureus, B. cereus, P. aeruginosa and B. subtilis strains N146, ST 1/III, Č13 and QST ix 713 exhibit antifungal activity against Colletotrichum spp. in vitro and in vivo. S. hygroscopicus ensures fruit protection at the same level as chemical fungicides (trifloxistrobin, boscalid+pyraclostrobine, pyrimethanil+flukvinconazol, captan). Cultivation of S. hygroscopicus in the medium with 15.07 g/l glycerol, 5.28 g/l yeast extract and 0.81 g/l phosphates, for 3-4 days, ensures maximal antifungal activity against Colletotrichum spp. Antifungal activity of S. hygroscopicus is based on production of extracelular, thermostable metabolites. Two-month storage of treated apple fruits at 2±0.5°C does not reduce antifungal activity of S. hygroscopicus. Oregano and thyme essential oils exhibit strong inhibitory effects on Colletotrichum