Identification of Indigenous Bradyrhizobium japonicum Strains Isolated from Different Soil Types in Western Slavonia

Abstract

Proučavanje prirodne populacije Bradyrhizobium japonicum kao i selekcija najučinkovitijih sojeva za komercijalnu poizvodnju, nameće potrebu za brzim i pouzdanim metodama identifikacije. Glavni cilj ovih istraživanja je komparacija različitih metoda za identifikaciju autohtonih sojeva B. japonicum izoliranih iz različitih tipova tala zapadne Slavonije. Fiziološki testovi, ispitivanje otpornosti na antibiotike, SDS-PAGE (sodium dodecil sulfat poliakrilamid gel elektroforeza) ukupnih staničnih proteina i RAPD (random amplified polymorphic DNA) analiza, korišteni su za identifikaciju sojeva i utvrđivanje varijabilnosti unutar prirodne populacije B. japonicum. Rezultati svake navedene metode prevedeni su u dvodimenzionalnu binarnu matricu i nakon analize podataka primjenom biostatističkog NTSYS programa, dobiven je dendrogram koji pokazuje relativnu sličnost između istraživanih sojeva. Na osnovi rezultata svih primjenjenih metoda utvrđeno je da je RAPD analiza najosjetljivija i najpouzdanija za identifikaciju sojeva. Najmanje razlike između B. japonicum sojeva utvrđene su ispitivanjem otpornosti na antibiotike. Na osnovi rezultata svih metoda, sa iznimkom prirodne otpornosti na antibiotike, utvrđeno je da se izolati bitno razlikuju od referentnih sojeva iz komercijalnog inokuluma te se mogu smatrati autohtonim za to područje. Proteinski i RAPD profili jasno su ukazali na postojanje dvaju bitno različitih grupa sojeva unutar prirodne populacije.Fast and reliable strain identification methods are needed for studying Bradyrhizobium japonicum field population as well as for the selection the most efficient strains for commercial inoculant production. The main aim of the present investigation was to compare different methods for identification of indigenous B. japonicum strains isolated from different soil types in western Slavonia. Physiological tests, intrinsic antibiotic resistance, SDS-PAGE (sodium dodecil sulphate polyacrilamide gel electrophoresis) of total cell proteins and RAPD (random amplified polymorphic DNA) analysis were used for strain identification and the assessment of variability within natural B. japonicum population. The results of each method were converted to two-dimensional binary matrix and dendrograms, showing relative similarity among B. japonicum strains, were obtained by using biostatistical NTSYS programe. The results showed that among all the methods used in this work, that RAPD analysis was the most sensitive and reliable for strain identification. The lowest level of differentiation among B. japonicum strains were determined by using intrinsic antibiotic resistance. All the methods used in this work, with the exception of intrinsic antibiotic resistance, revealed that all field isolates essentially differed from commercial strains and so can be regarded as indigenous strains for that area. Protein and RAPD profiles very clearly indicated that among natural field population two highly divergent group of strains can be determined

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