master's thesis
Molecular identification of fungi from families Diaporthaceae and Botryosphaeriaceae isolated from wood of figs in Croatia

Sanja Marin (2016)
University of Zagreb
Faculty of Agriculture
Department of Plant Pathology
Metadata
TitleMolekularna identifikacija gljiva iz porodica Diaporthaceae i Botriosphaeriaceae izoliranih iz drva smokve u Hrvatskoj
AuthorSanja Marin
Mentor(s)Tihomir Miličević (thesis advisor)
Abstract
Mikoze drva smokve (Ficus carica L.) su biljne bolesti koje uzrokuju fitopatogene gljive. Do danas u Hrvatskoj bolesti drva smokve nisu istraživane, dok su u svijetu ta istraživanja ukupno gledano relativno rijetka. Od gljiva patogena drva smokve najvažnijima se smatraju one iz porodice Diaporthaceae, no u posljednje vrijeme u svijetu gljive iz porodice Botryosphaeriaceae dobivaju na važnosti kao novi patogeni smokve. S ciljem utvrđivanja prisutnosti i taksonomske pripadnosti gljiva iz porodica Diaporthaceae i Botryosphaeriaceae na smokvi u Hrvatskoj, s više lokaliteta na području Dalmacije tijekom 2014. i 2015. godine prikupljeni su uzorci bolesnog drva smokve. Iz uzoraka su izolirani izolati gljiva u čistoj kulturi, a oni koje su prema fenotipskim obilježjima pripadali u porodicu Diaporthaceae ili Botryosphaeriaceae izdvojeni su od ostalih izolata te podvrgnuti molekularnoj analizi. U navedenoj analizi svim navedenim izolatima izolirana je ukupna genomska DNA, a potom je metodom lančane reakcije polimerazom (PCR), za izolate iz obiju porodica, umnožen molekularni marker ITS korištenjem para početnica ITS5/ITS4, odnosno za izolate iz porodice Botryosphaeriaceae dodatno još i molekularni marker EF1-α korištenjem para početnica EF1-728F/EF1-986R. Dobiveni PCR produkti umnažanja molekularnih markera pročišćeni su uporabom kita za pročišćavanje PCR produkata, te je sekvenciranjem utvrđen nukleotidni slijed njihovih sekvenci. Sve sekvence uspoređene su s odgovarajućim sekvencama referentnih izolata u međunarodnoj banci gena GenBank korištenjem metode BLAST te dodatno i filogenetskom analizom. Navedenim metodama identificirane su sljedeće vrste iz porodice Botryosphaeriaceae: Neofusicoccum parvum, Dothiorella omnivora, Dothiorella sempervirentis, Spencermartinsia viticola te Spencermartinsia plurivora. Jedinom izoliranom izolatu gljive iz porodice Diaporthaceae nije uspješno sekvenciran molekularni marker ITS te je stoga, prema morfološkim karakteristikama kolonije na KDA, identificiran do razine roda kao Diaporthe sp. Donesen je zaključak da, suprotno očekivanom, gljive iz porodice Botryosphaeriaceae na smokvi u Hrvatskoj imaju znatno veću zastupljenost od vrsta iz porodice Diaporthaceae. Također, od pet vrsta gljiva iz porodice Botryosphaeriaceae prvi put u svijetu nađenih na smokvi, čak dvije, N. parvum i S. viticola bi mogli biti potencijalno važni patogeni na smokvi.
KeywordsFicus carica molecular identification wood mycoses Botryosphaeriaceae Diaporthaceae
Parallel title (English)Molecular identification of fungi from families Diaporthaceae and Botryosphaeriaceae isolated from wood of figs in Croatia
Committee MembersTihomir Miličević (committee chairperson)
Boris Duralija (committee member)
Aleksandar Mešić (committee member)
Joško Kaliterna (committee member)
GranterUniversity of Zagreb
Faculty of Agriculture
Lower level organizational unitsDepartment of Plant Pathology
PlaceZagreb
StateCroatia
Scientific field, discipline, subdisciplineBIOTECHNICAL SCIENCES
Agronomy
Study programme typeuniversity
Study levelgraduate
Study programmePhytomedicine
Academic title abbreviationmag. ing. agr.
Genremaster's thesis
Language Croatian
Defense date2016-09-27
Parallel abstract (English)
Mycoses of wood of fig (Ficus carica L.) are plant diseases caused by phytopathogenic fungi. To date in Croatia diseases of wood of figs have not been investigated, while worldwide, researches on this topic are overall relatively rare. Among fungi which are wood pathogens of figs, the most important ones are considered those from the family Diaporthaceae, but lately fungi from the family Botryosphaeriaceae are gaining importance as new pathogens of figs. In order to determine their presence and identify fungi from families Diaporthaceae and Botryosphaeriaceae on fig trees in Croatia, during the 2014 and 2015 samples of diseased wood of figs were collected from several locations in Dalmatia. From the samples, isolates of fungi were obtained in pure cultures, and those who have to phenotypic characteristics typical to the families Diaporthaceae or Botryosphaeriaceae were separated from other isolates and subjected to molecular analysis. The analysis included isolation of genomic DNA followed by polymerase chain reaction (PCR) amplification of molecular markers ITS (for isolates from both families) using primer pair ITS5 / ITS4, and EF1-α (for isolates from family Botryosphaeriaceae) using primer pair EF1-728F / EF1-986R. The PCR amplification products were purified using a kit for the purification of PCR products, and their nucleotide sequences were determined by DNA sequencing. All the sequences were compared with corresponding reference sequences in the international gene database GenBank using BLAST and by phylogenetic analysis. The following species were identified, belonging to the family Botryosphaeriaceae: Neofusicoccum parvum, Dothiorella omnivora, Dothiorella sempervirentis, Spencermartinsia viticola and Spencermartinsia plurivora. The only isolate in this study belonging to the family Diaporthaceae didn’t have its molecular marker ITS successfully sequenced and therefore, according to the morphological characteristics of its colonies on PDA, it was only identified to the genus level as Diaporthe sp.. It was concluded that, opposite to the expectations, the fungi from the family Botryosphaeriaceae have significantly greater prevalence on fig trees in Croatia than those from the family Diaporthaceae. Also, among the five species of fungi from the family Botryosphaeriaceae for the first time in the world found on fig trees in this study, even two species, N. parvum and S. viticola, may potentially be important pathogens of fig.
Parallel keywords (Croatian)Ficus carica molekularna identifikacija mikoze drva Botryosphaeriaceae Diaporthaceae
Resource typetext
Access conditionOpen access
Terms of usehttp://rightsstatements.org/vocab/InC/1.0/
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:204:431568
CommitterValentina Bezek