Isolation and characterization of endophytic bacteria from soybean (Glycine max L.)

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.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