The Formulation of Tow Media for the Selective Isolation of Agrobacterium Species from Soils

Abstract

Ten isolates (Tu1, Tu2,…, Tu10) of the plant pathogenic bacteria, Agrobacterium tumefaciens were isolated from the stem tumours of Launaea capitata, Rosa involucrata and Jasminum sp.. The isolates were authenticated using Helianthus annus seedlings as a host of authentication. Nine of the Tu isolates were found to be pathogenic whereas Tu9 was considered as a non- pathogenic agrobacteria. These isolates were considered and used as standard Agrobacterium tumefaciens isolates throughout this study. In order to formulate two selective media (one for the selective isolation of A. tumefaciens and the other for A. rhizogenes) minimum inhibitory concentrations (MICs) of selenite, tolerance to salinity and susceptibilities to certain antibotics were determined. The MICs of the isolates to selenite ranged from 4 to 14 g/ l. However, all of the Tu isolates were able to tolerate high concentrations of NaCl (2.5%), and were all sensitive to Streptomycin. Considering this, two selective media Yeast Arabitol- Selenite medium (YAS) and Yeast Erythrytol- Selenite medium (YES) were formulated for the selective isolation of A. tumefaciens and A. rhizogenes, respectively. Sixty isolates were recovered from soil samples on different media as follows: twenty on Yeast Mannitol- Selenite medium (YMA) (SYMS1, SYMS2,….SYMS20), 20 on YAS medium (SYAS1, SYAS2,…SYAS20) and 20 on YES medium (SYES1, SYES2,…SYES20). These soil isolates were characterized using cultural, microscopical and biochemical characteristics. The isolates were found to be Gram- negative, rods, motile and non- endospore forming and were thus considered as Agrobacterium isolates. SYAS isolates which were unable to utilize erythritol but could utilize arabitol were identified as presumptive A. tumefaciens. On the other hand, all SYES isolates were able to utilize erythritol as a sole carbon source and were considered as presumptive A. rhizogenes. SYMS isolates gave variable results indicating the non- selectivity of the YMS medium. The results of MICs of selenite ranged from 4- 14 g/ l for all isolates while the MICs of tellurite ranged from 0.08- 1.28 g/ l for SYMS isolates, from 0.08- 0.32 g/ l for SYAS isolates and from 0.64- 1.28 g/ l for SYES isolates. So, if tellurite is added to YAS and YES media in specific concentrations, it will improve the selectivity of these media. All of the isolates were tested for the presence or absence of Ti or Ri plasmids using VCR and VCF pathogenicity primers. Nine of the Tu, seven of the 20 SYMS, five of the 20 SYAS and eight of the 20 SYES isolates were found to harbor Ti or Ri plasmids. To determine the type of plasmid, tumourigenicity tests were conducted for all of the pathogenic isolates. Three of the pathogenic SYMS isolates and all of the five pathogenic SYAS isolates and none of the pathogenic SYES isolates were found to be tumourigenic and hence were considered as A. tumefaciens. Pathogenic soil isolates which gave negative results for tumourigenicity tests were tested for rhizogenicity usin Boswellia papyrifera explants. All of these isolates were able to induce hairy- root formation. They were then considered as A. rhizogenes. However, the non- pathogenic isolates were considered as A. radiobacter. The results of rhizogenicity tests indicated that rooting of Boswellia papyrifera could be much improved by inoculating the explants with Agobacterium rhizogenes. This may contribute to the establishment of a successful tissue culture protocol for the regeneration of Boswellia papyrifera and any other difficult- rooting plants