Izolacija i karakterizacija endofitskih bakterija iz soje (Glycine max L.)

master's thesis

• Title (english): Isolation and characterization of endophytic bacteria from soybean (Glycine max L.)
• Author: Dora Krznarić
• Mentor: Sanja Sikora (mentor)
• Committee member: Sanja Sikora (predsjednik povjerenstva)
• Committee member: Milan Poljak (član povjerenstva)
• Committee member: Klaudija Carović-Stanko (član povjerenstva)
• Committee member: Sanja Kajić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Agriculture; (Department of Microbiology); Zagreb
• Defense date and country: 2018-09-28, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Agronomy

Abstract

Endofitske bakterije koloniziraju unutrašnjost biljnih tkiva, a pritom ne uzrokuju
negativne posljedice. Glavni cilj ovih istraživanja je genotipska i fenotipska
karakterizacija endofitskih bakterija izoliranih iz različitih sorti soje. Izolirano je
ukupno 29 endofita iz tri različita tkiva – korijena, kvržica i stabljike. Genotipska
karakterizacija uključila je primjenu sekvenciranja 16S rRNA (rrs) gena. Pomoću
navedene molekularne metode identificirane su bakterijske vrste i utvrđena je
genetska raznolikost unutar populacija endofitskih bakterija na soji. Identificirano je
pet skupina bakterija različitih rodova: Pseudomonas spp., Sphingomonas spp.,
Bradyrhizobium spp., Rhizobium spp. i Agrobacterium spp. Od 29 izolata, najveća
pripadnost jednoj vrsti pripada skupini roda Pseudomonas spp. Svi izolati koji su
identificirani kao B. japonicum, izolirani su iz korijena i kvržice biljke. Dokazana je
uska povezanost rodova Rhizobium i Agrobacterium. Fenotipska karakterizacija
sojeva prikazala je raznolikost i otpornost endofitskih bakterija. Većina izolata je
Gram negativna, štapićastog oblika, bez mogućnosti stvaranja kapsule. Dokazana je
varijabilnost fenotipskih karakteristika izolata, ali i otpornost prema nepovoljnim
uvjetima poput povišene temperature, povećanog sadržaja soli te niske i visoke pH
vrijednosti. Prilikom ispitivanja izolata za detekciju fluorescentnog pigmenta
dokazano je kako samo bakterije vrste Pseudomonas produciraju pigment pod UV
svjetlom. Većina sojeva pokazala je tolerantnost na istraživane antibiotike. Većina
katalaza pozitivnih izolata pripadaju unutar rodova Bradyrhizobium ili Rhizobium,
dok su katalaza negativni izolati identificirani kao Pseudomonas, Sphingomonas i
Agrobacterium spp.. Dokazano je kako 85% ispitanih izolata ne reagira na enzim
oksidazu, dok oni koji reagiraju pripadaju vrsti Pseudomonas spp. Potrebna je daljnja
karakterizacija endofita kako bi se utvrdio njihov utjecaj na razvoj biljke.

Abstract (english)

Endophytic bacteria colonize the inside of the tissue without causing any negative
effects. The main aim of this study is the genotypic and phenotypic characterization of
endophyitic bacteria isolated from different soybean cultivar. A total of 29 endophytes
were isolated from three different tissues – root, nodule and stem. Genotypic
characterization includes using of sequencing 16S rRNA (rrs) genes. Bacterial species
were identified by using this molecular method and genetic diversity was determined
within the population of endophztic bacteria of the soybean. Five different genera of
bacteria were identified: Pseudomonas spp., Sphingomonas spp., Bradyrhizobium
spp., Rhizobium spp., and Agrobacterium spp. From a total of 29 endophytes, most
strains were identified as Pseudomonas spp. All isolates identified as B. japonicum
were isolated from the roots and nodules of the plant. The close assocation beetwen
Rhizobium and Agrobacterium species is proven. The phenotypic characterization of
strains shows diversity and resistance of endophytes. Most of the isolates are Gram
negative, rod-shaped, without capability of capsule production. The variability of the
phenotypic characteristics of isolates was demonstrated, as well as the resistance to
adverse conditions such as elevated temperature, increased salt content, and low and
high pH values. During the detection of fluorescent pigment, it has been shown that
only Pseudomonas species produce pigment under UV light. Most strains showed
tolerance to the specific antibiotics. Most of the catalase positive isolates belong to
Bradyrhizobium or Rhizobium spp., while the catalase negative isolates belong to the
Pseudomonas, Sphingomonas and Agrobacterium spp. It was shown that 85% of the
tested isolates do not respond to the enzyme oxidase while those that react to the
enzyme belongs to the Pseudomonas spp. Further characterization of endophytes is
needed to determine their influence on plant development.

• Keywords: endofitske bakterije; soja (Glycine max L.); sekvenciranje 16S rRNA gena; fenotipska karakterizacija
• Keywords (english): endophytic bacteria; soybean (Glycine max L.); sequencing of 16S rRNA gene; phenotypic characterization
• Language: croatian
• URN:NBN: urn:nbn:hr:204:332404
• Study programme: Title: Agroecology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/ magistra inženjer/inženjerka agroekologija (magistar/ magistra inženjer/inženjerka agroekologija)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Repository: Repository Faculty of Agriculture University of Zagreb
• Created on: 2018-11-05 08:25:02