Identifikacija bakterija stimulatora biljnog rasta iz rodova Bradyrhizobium i Bacillus

Abstract

Uprkos variranju osobina stumilsanja rasta biljaka (PGP) kod razliĉitih vrsta, većina bakterijskih sojeva koji ispoljavaju višestruka PGP svojstva moţe biti korisna u formulaciji novih inokulanata. Istraţivanje efektivnih sojeva, pre PGP karakterizacije, zahteva izolaciju i identifikaciju odgovarujućih vrsta bakterija koje stimulišu biljni rast (PGPB). Cilj ovih istraţivanja bio je izolacija i molekularna identifikacija PGPB. Bakterije iz roda Bradyrhizobium izolovane su iz korenskih kvrţica razliĉitih sorti soje. Bakterije koje pripadaju rodu Bacillus izolovane su iz rizosfernog, poljoprivrednog i nepoljoprivrednog zemljišta. Svi uzorci za izolaciju PGPB prikupljeni su s razliĉitih lokaliteta u Vojvodini. Karakterizacija izolata izvršena je na osnovu morfoloških i biohemijskih svojstava. Izolati koji su pokazali sliĉnost sa vrstama Bradyrhizobium sp. and Bacillus sp. bili su predmet molekularne karakterizacije. Nakon ekstrakcije DNK, parcijalna 16S rDNA genska sekvenca iz izolata je umnoţena PCR metodom upotrebom univerzalnih prajmera fD1 i rP3. Preĉišćavanje i sekvencioniranje umnoţenih fragmenata uraĊeno je u Macrogen, Juţna Koreja. Sekvence su analizirane pomoću programa FinchTV i izvršeno je višestruko poreĊenje dobijenih sekvenci sa postojećim sekvencama u NCBI GenBank bazi podataka. Svi izolati Bradyrhizobium identifikovani su kao B. japonicum. U okviru Bacillus izolata, identifikovano je devet razliĉitih vrsta (B. megaterium, B. cereus, B. pumilus, B. subtilis, B. thuringiensis, B. safensis, B. mycoides, Lysinibacillus fusiformis, and L. sphaericus). Nastavak karakterizacije ovih izolata preko PGP skrininga neophodan je za odabir potencijanlo efektivnih sojeva za inokulaciju u kontrolisanim i poljskim uslovima.

Despite the variability within the plant growth promoting properties (PGP) in different species, most of bacterial strains that exhibit multiple PGP traits might be useful in formulating new inoculants. Search for effective strains, prior to PGP characterization, require isolation and identification of desirable species of plant growthpromoting bacteria (PGPB). The aim of this study was to isolate and identify PGPB on the basis of molecular characteristics. Bacteria of Bradyrhizobium genus were obtained from root nodules of different soybean cultivars. Bacteria belonging to genus Bacillus were isolated from different soil samples which included the rhizosphere of plants, agricultural and non-agricultural soils. All samples for PGPB isolation were collected from various parts of the Vojvodina Province. Characterization of isolates was performed according to morphological and biochemical properties. Isolates showing resemblance with Bradyrhizobium sp. and Bacillus sp. were subjected to molecular characterization. Following DNA extraction, a partial 16S rDNA gene sequence from the isolates was amplified by PCR using universal primers fD1 and rP3. Purification and sequencing of the amplified fragments were done in the Macrogen, South Korea. Sequences were analyzed using the program FinchTV and compared to sequences in NCBI GenBank for identification. All isolates of Bradyrhizobium were identified as B. japonicum. Within Bacillus isolates, nine different species (B. megaterium, B. cereus, B. pumilus, B. subtilis, B. thuringiensis, B. safensis, B. mycoides, Lysinibacillus fusiformis, and L. sphaericus) were identified. Further characterization of these isolates through PGP screening will be necessary to select potentially effective strains for inoculation assays in greenhouse and field.