Identifikacija gliv, ki bi lahko povzročale zgodnje odpadanje plodov oljke, na osnovi črtne kode DNA

Premrl, Petja (author), Dolinar, Marko (mentor), Tavčar Verdev, Petra (comentor)

V oljkarstvu severnega Sredozemlja se v zadnjih letih pojavlja problem odpadanja plodov oljk v zgodnji fazi razvoja. Plodovi potemnijo, se posušijo in odpadejo, kar lahko pridelek zmanjša za več kot polovico. Razlog za ta pojav še ni znan. Različni viri kot vzrok navajajo abiotske dejavnike, kot sta suša in temperaturna nihanja, spet drugi pa navajajo prisotnost patogenih gliv ali škodljivih insektov. Cilj diplomskega dela je bil z uporabo črtne kode DNA identificirati vrste gliv, ki so prisotne na okuženih plodovih oljk in bi lahko povzročile njihovo zgodnje odpadanje. Izolirali smo genomsko DNA in z verižno reakcijo s polimerazo (PCR) pomnožili dele genoma, ki predstavljajo vmesnik – regijo ITS – med zapisoma za ribosomsko RNA 18 S in 28 S. Regija ITS velja za najprimernejše variabilno zaporedje v genomu gliv, ki omogoča določitev organizma do vrste natančno. Dobljenim produktom PCR smo določili nukleotidno zaporedje. S primerjavo teh zaporedij z zaporedji iz zbirk GenBank in BOLD smo identificirali več rodov gliv, med katerimi najdemo tudi znane patogene glive, ki okužujejo oljke. Najpogosteje identificirani rodovi gliv so bili Alternaria, Aureobasidium, Cladosporium, Neofusicoccum in Trichoderma. Določanje gliv do vrste natančno se je izkazalo za problematično, saj smo pri analizi posameznih zaporedij v zbirkah pogosto naleteli na več zadetkov z visoko podobnostjo, ki so pripadali različnim vrstam istega rodu. Mnogi rodovi vsebujejo poleg patogenih tudi endofitne glive, kot sta Alternaria alternata in Aureobasidium pullulans, ki lahko pod določenimi pogoji (npr. ob temperaturnem nihanju) postanejo patogene za oljke. Naši rezultati kažejo, da je prezgodnje odpadanje plodov oljk najverjetneje posledica okužbe z eno ali več glivami, katerih zaporedja smo našli v vzorcih okuženih plodov oljke. Možno pa je tudi, da so za okužbo krivi abiotski dejavniki, ki vplivajo na nekatere endofitne glive, da postanejo patogene.

• Language: Slovenian
• Keywords: oljka, identifikacija gliv, črtna koda DNA, regija ITS
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2024
• PID: 20.500.12556/RUL-161072
• COBISS.SI-ID: 213396227
• Publication date in RUL: 06.09.2024

Secondary language

• Language: English
• Title: Identification of fungi which could cause early fruit drop of olives based on DNA barcoding

Abstract

In recent years, olive cultivation in the northern Mediterranean has faced the problem of olive fruit drop, occurring at an early stage of fruit development. The fruits darken, dry out, and fall off, which can reduce the yield by more than half. The cause of this phenomenon is still unknown. Various sources cite abiotic factors, such as drought and temperature fluctuations as the cause, while others suggest the presence of pathogenic fungi or harmful insects. The aim of this thesis was to identify the species of fungi present on infected olive fruits which could cause early fruit drop using, DNA barcoding. We isolated genomic DNA and used polymerase chain reaction (PCR) to amplify genome regions that represent the internal transcribed spacer (ITS) region between the 18 S and 28 S ribosomal RNA genes. The ITS region is considered the most suitable variable sequence in the fungal genome allowing for accurate identification of the organism to the species level. We determined the nucleotide sequences of the obtained PCR products. By comparing these sequences with those in the GenBank and BOLD databases, we identified several fungal genera, including known olive pathogens. The most frequently identified fungal genera were Alternaria, Aureobasidium, Cladosporium, Neofusicoccum, and Trichoderma. Identifying fungi to the species level proved problematic, as the analysis of individual sequences often revealed multiple high-similarity hits belonging to different species of the same genus. Many genera, in addition to pathogenic species, also contain endophytic fungi, such as Alternaria alternata and Aureobasidium pullulans, which can become pathogenic to olives under certain conditions (e.g., temperature fluctuations). Our results suggest that premature olive fruit drop is most likely due to infection by one or more fungi whose sequences we found in samples of infected olive fruits. However, it is also possible that abiotic factors are responsible for the infection as they promote the pathogenicity of certain endophytic fungi.

• Keywords: olive tree, identification of fungi, DNA barcoding, ITS region