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KARAKTERIZACIJA VRSTA RODA Fusarium PATOGENA SIRKA [Sorghum Bicolor (L.) Moench] U SRBIJI I UTVRĐIVANJE OSETLJIVOSTI GENOTIPOVA

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KARAKTERIZACIJA VRSTA RODA Fusarium PATOGENA SIRKA [Sorghum Bicolor (L.) Moench] U SRBIJI I UTVRĐIVANJE OSETLJIVOSTI GENOTIPOVA UNIVERZITET U BEOGRADU POLJOPRIVREDNI FAKULTET mr Danijela T. Ristić KARAKTERIZACIJA VRSTA RODA Fusarium PATOGENA SIRKA [Sorghum bicolor (L.) Moench] U SRBIJI I UTVRĐIVANJE OSETLJIVOSTI GENOTIPOVA doktorska disertacija Beograd, 2012. UNIVERSITY OF BELGRADE FACULTY OF AGRICULTURE Danijela T. Ristić, MsC CHARACTERIZATION OF Fusarium SPECIES PATHOGEN FOR SORGHUM [Sorghum bicolor (L.) Moench] IN SERBIA AND GENOTYPE SUSCEPTIBILITY Doctoral Dissertation Belgrade, 2012 Komisija za ocenu i odbranu: Mentor: dr Aleksandra Bulajić, vanredni profesor Univerzitet u Beogradu−Poljoprivredni fakultet Članovi komisije: dr Mirko Ivanović, redovni profesor Univerzitet u Beogradu−Poljoprivredni fakultet dr Janoń Berenji, naučni savetnik Institut za ratarstvo i povrtarstvo, Novi Sad dr Branka Krstić, redovni profesor Univerzitet u Beogradu−Poljoprivredni fakultet dr Jelena Lević, naučni savetnik Institut za kukuruz „Zemun Polje“, Beograd Datum odbrane: _____________________ Posebnu zahvalnost želim da izrazim svom mentoru, dr Aleksandri Bulajić, vanr. prof. koja mi je pružila neizmernu podršku, znanje i iskustvo, ne samo u izradi doktorske disertacije, već uopšte u naučnom radu. Zbog velikodušne mudrosti i neprocenjive pomoći i podrške veliku zahvalnost dugujem dr Branki Krstić, red. prof. Zahvaljujem i dr Janošu Berenjiju, naučnom savetniku na pomoći i korisnim savetima tokom realizacije eksperimentalnog dela i završne verzije teksta. Posebnu zahvalnost dugujem dr Mirku Ivanoviću, red. prof. i dr Jeleni Lević, naučnom savetniku na korisnim savetima i sugestijama koji su doprineli poboljšanju kvaliteta završne verzije teksta. Zahvalnost dugujem mr Dragosavu Mutavdžiću, istraživaču saradniku za pruženu pomoć pri obradi statističkih podataka. Takođe, želim da izrazim svoju zahvalnost dr Ivani Stanković, doc., Ani Vučurović, dipl. inž., Katarini Milojević, dipl. inž., Dušanu Nikoliću, dipl. inž. i Dragani Đurić, teh. saradniku zbog njihove podrške i pomoći koju su pružili mom istraživanju i stvaranju ove disertacije. Beskrajno sam zahvalna svojoj porodici, koja je uvek uz mene i čija ljubav i podrška ne znaju za granice. KARAKTERIZACIJA VRSTA RODA Fusarium PATOGENA SIRKA [Sorghum bicolor (L.) Moench] U SRBIJI I UTVRĐIVANJE OSETLJIVOSTI GENOTIPOVA Rezime. Tokom trogodińnjeg perioda od 2009. do 2011. godine, pregledom četiri lokaliteta gajenja sirka u Vojvodini, zabeleņena je pojava intenzivnih simptoma truleņi korena i stabla. Intenzitet zaraze useva sirka, bio je veoma visok, a suńenje i brzo propadanje zaraņenih biljaka rezultiralo je procenjenim gubicima od preko 90%. Sa ciljem da se dobije uvid u prisustvo vrsta roda Fusarium i tako rasvetli etiologija oboljenja u Srbiji, obavljena je identifikacija na osnovu proučavanja morfolońkih svojstava i molekularne karakterizacije izolata dobijenih iz zaraņenih biljaka u polju i semena. Na osnovu obavljenih istraņivanja ustanovljeno je da odabrani izolati pripadaju vrstama roda Fusarium: F. equiseti, F. proliferatum, F. graminearum i F. thapsinum, kao i vrsti Epicoccum nigrum. Proučavana svojstva ispitivanih izolata analizirana su kao mogući taksonomski kriterijum za razlikovanje vrsta roda Fusarium i preporučuje se korińćenje najmanje dva taksonomska kriterijuma i to morfolońkih svojstava i molekularne karakterizacije. Analize nukleotidnih sekvenci pet genomnih regiona, DNK iz ITS regiona, gena koji kodiraju elongacioni faktor TEF−1α, β−tubulin i calmodulin, kao i mitohondrijalne male podjedinice ribozomske DNK, pokazale su različite potencijale za preciznije razdvajanje vrsta roda Fusarium. Rekonstrukcijom filogenetskih stabala dat je doprinos u rasvetljavanju evolutivne meĎupovezanosti različitih vrsta, kao i izolata u okviru jedne vrste i pruņilo je uvid u prisustvo i rasprostranjenost vrsta Fusarium u nańoj zemlji. Ispitivanja u okviru ove disertacije ukazala su na postojanje različitih vidova osetljivosti komercijalnih sorti i oplemenjivačkog materijala in vitro i u poljskim ogledima u uslovima prirodne zaraze i veńtačke inokulacije sa prevalentnim vrstama F. equiseti i F. proliferatum. Dobijeni rezultati dalje će se koristiti u programima oplemenjivanja gajenog sirka na otpornost. Ključne reči: gajeni sirak, fuzariozna truleņ, taksonomski kriterijumi, osetljivost genotipova Naučna oblast: Biotehničke nauke Uţa naučna oblast: Fitopatologija UDK: 632.95:633.17(043.3) CHARACTERIZATION OF Fusarium SPECIES PATHOGEN FOR SORGHUM [Sorghum bicolor (L.) Moench] IN SERBIA AND GENOTYPE SUSCEPTIBILITY Abstract. Intensive symptoms of root and stem rot of sorghum were recorded in four sorghum growing localities in the province of Vojvodina, during a three year investigation period from 2009 to 2011. Estimated loss of inspected crops was over 90%, because of drying and deterioration of infected sorghum plants in highly infected crops. In order to get better insight into the presence of Fusarium species and to clarify etiology of the disease in Serbia, the identification was carried out based on the study of morphological properties and molecular characterization of isolates originating from infected plants in the field and seeds. On the basis of the investigations it was found that the selected isolates belong to the species of the genus Fusarium: F. equiseti, F. proliferatum, F. graminearum and F. thapsinum, as well as, to the species Epicoccum nigrum. Studied properties of selected isolates were analyzed as a possible taxonomic criterion for distinguishing species of the genus Fusarium. Utilization of at least two taxonomic criteria in particular/as follows morphological properties and molecular characterization are recommended. Nucleotide sequences analysis of five genomic regions of DNA from the ITS region, genes encoding elongation factor TEF−1α, β−tubulin and calmodulin, as well as, the mitochondrial small subunit ribosomal DNA showed different potentials for delineation of Fusarium species. Contribution in clarifying evolutionary interlinkage/interconnections of different species, and strains within a species was given by reconstruction of phylogenetic trees which provided insight into the presence and distribution of Fusarium species in our country. The existence of various forms of susceptibility of commercial varieties and breeding materials were revealed, both in vitro and in field experiments, to natural infection and artificial inoculation with F. equiseti and F. proliferatum. Results obtained in this study will contribute in the deployment of sorghum breeding programs for resistance to these important pathogens. Keywords: cultivated sorghum, fusarious rot, taxonomic criteria, genotype susceptibility Scientific field: Biotechnical Science Scientific discipline: Phytopathology UDC: 632.95:633.17(043.3) SADRŢAJ 1. UVOD…………………………………………………………………………….. 1 2. PREGLED LITERATURE…………………………………………………….. 3 2.1. Privredni značaj i botanička klasifikacija gajenog sirka………........................... 3 2.2. Najznačajnije agronomske forme gajenog sirka……........................................... 5 2.3. Bolesti gajenog sirka u svetu................................................................................ 7 2.4. Bolesti gajenog sirka u Srbiji................................................................................ 11 2.5. Gljive roda Fusarium i njihov značaj................................................................... 12 2.5.1. Značajne vrste roda Fusarium opisane na gajenom sirku u svetu…................. 13 2.5.2. Značajne vrste roda Fusarium opisane na gajenom sirku u Srbiji..................... 14 2.6. Klasifikacija i taksonomija roda Fusarium........................................................... 16 2.7. Najznačajnije Fusarium vrste kao patogeni gajenog sirka................................... 20 3. CILJEVI ISTRAŢIVANJA……………………………………………………. 27 4. MATERIJAL I METOD RADA………………………………………………. 28 4.1. Sakupljanje uzoraka obolelih biljaka………........................................................ 28 4.2. Izolacija patogena………..................................................................................... 28 4.3. Dobijanje čistih kultura monosporijalnih izolata i njihovo čuvanje……............. 29 4.4. Provera patogenosti i izbor izolata za dalja ispitivanja…………......................... 30 4.5. Morfolońka svojstva anamorfa………….............................................................. 32 4.6. Morfolońka svojstva teleomorfa (peritecija)………............................................. 34 4.7. Molekularna detekcija i identifikacija………...................................................... 34 4.7.1. Ekstrakcija DNK…………............................................................................... 35 4.7.2. Lančana reakcija polimeraze (PCR)…….......................................................... 38 4.7.3. Vizuelizacija i analiza produkata PCR reakcija……......................................... 40 4.7.4. Prečińćavanje PCR produkta i sekvencioniranje…............................................ 41 4.7.5. Molekularna identifikacija i karakterizacija…................................................... 42 4.8. Ocenjivanje nivoa osetljivosti različitih genotipova sirka prema Fusarium spp….................................................................................................................... 44 4.8.1. Ocenjivanje nivoa osetljivosti klijanaca sirka prema Fusarium spp. u uslovima staklenika…........................................................................................ 44 4.8 4.8.2. Ocenjivanje nivoa osetljivosti stabla sirka prema Fusarium spp. u poljskim uslovima............................................................................................................
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