Isolation and Characterization of Agrobacterium tumefaciens Strains from Malakander Farm, University of Agriculture, Peshawar

Soil samples were collected from Agricultural University field. Five samples were collected from topsoil and subsoil. The samples were collected in a sterile polythene bag with the help of sterile spatula. The samples were brought to the laboratory and kept at 28°C for further use.

Soil stock solution was made by mixing 10 g soil with 90 ml saline solution. Serial dilutions of the soil sample were made. Soil solution sample (0.1 ml) was inoculated onto Yeast Extract Mannitol Agar (YEMA) medium (Mannitol 10g/l, Yeast extracts 1g/l, Dipotassium phosphate 0.5g/l, sodium chloride 0.2g/l, CaCl2 0.2g/l, MgSO4.7H2O 0.2g/l, FeCl3 0.01g/l, Agar 15g/l, pH 7.0). Petri dishes were incubated upside down for 48 h at 28°C. Pure cultures were isolated and bacteria were preserved in glycerol cultures.

Isolated bacteria were identified as A. tumefaciens using Gram staining and biochemical tests like Benedict test for production of 3-ketolactose, catalase, oxidase, citrate, motility and 2% NaCl tolerance tests.

For pathogenicity test potato (Solanum tuberosum) and carrot (Daucus carota) discs were used for tumour induction properties of Agrobacterium. Potato tubers and carrot roots were washed to remove soil. Both were cut into small slices on a sterile marble tile with the help of sterile knife under sterile conditions. The discs were sterilized with 15% commercial bleach for 15 min. The discs were then rinsed with sterile water three times. The discs were placed on Murashige and Skoog medium. Each disc was inoculated with 100 µl inoculum. The plates were sealed with parafilm and incubated in growth chamber 25°C for 3 weeks. After 3 weeks, the discs were checked for tumours development.

Colonies on YEM medium were observed as pink, pinkish red and brick red in colour. These colonies were circular in shape. The isolated bacteria (7 isolates) were rod shaped and red in colour (Gram negative) as given in Table I. Yellow ring was observed around the colony in about 1-2 h when 3-ketolactose production occurred. All the seven isolates were positive for 3-ketolactose production (Table I). In citrate test, growth was observed on the slant surface and the medium colour was changed from green to dark greenish blue colour indicating positive results (Table I). Dispersed growth was observed from line of inoculation in motility test (Table I). Black precipitate was also observed, indicating H2S production. The selected seven isolates were positive for H2S production (Table I). The isolated bacteria were positive for 2% salt in the growth medium (Table I). In catalase test, bubbles were formed on the slide after adding H2O2. The isolated bacteria were positive for catalase test (Table I). The inoculated filter paper turned violet immediately after adding the reagent in oxidase test showing positive results (Table I).

All the isolates were positive for pathogenicity test under in vitro bio-assays (Table II). Tumours were developed around the central vascular stem.

Table I. Morphological and biochemical characteristics of Agrobacterium tumefaciens isolates (+ sign indicates positive result and – sign indicates negative result.

The aim of this project was to isolate and characterize A. tumefaciens strains from soil from Agricultural university field and to confirm their characteristics by morphological, biochemical and pathogenicity tests. On the basis of colour development, some pink colour colonies were observed on YEM agar medium as reported previously (Kumar et al., 2013). However, in our results pinkish red and brick red coloured colonies were also observed on YEM agar medium. The colonies were smooth, circular and translucent. The bacterial cells were rod shaped in Gram staining as described previously (Setti and Bencheikh, 2013). For further confirmation of A. tumefaciens, various biochemical tests were performed, and the isolates were found negative in Gram test and positive in motility, catalase, oxidase, 3-ketolactose production, H2S production and 2% salt tolerance.

The native soil bacterium, A. tumefaciens, is famous for plant diseases. A. tumefaciens initiates crown gall disease in many dicotyledonous plants, particularly in members of Rosaceae like almond, cherry, pear, peach, apple, roses and raspberry. Crown gall disease produces tumor like swellings (galls) that usually arise at the plant crown just above the ground. The bacterium transfers a piece of plasmid DNA into the plant through infection. This T-DNA incorporates into the genome of the plant and produces tumors in the host plant and alters the metabolism of the host plant. The infected plant produces new plant metabolites such as opines. Opines are specific by-products of amino acids that are used as a source of carbon and nitrogen by the tumor causing bacteria. Agrobacteria are famous for utilizing a broad range of substrates (Koivunen et al., 2004). The ability to form tumours by the selected isolates on potato and carrot confirmed them as pathogenic strains of A. tumefaciens as suggested previously (Sarker et al., 2011).

The isolates possessed pathogenic characteristics that indicate the disease-causing ability of the isolated A. tumefaciens strains. These virulence characteristics which are due to the presence of Ti plasmid in these strains are potential threats to cultivated crops and could be disseminated to other localities through transportation, human and farming practices, and germplasm exchange.

Current study confirmed the prevalence of pathogenic strains of A. tumefaciens in local soil samples. These findings highlight the need for proper management and control strategies to avoid or minimize crop damages in case of infections and prevent the spread of pathogenic strains of A. tumefaciens through quarantine measures.

The author has no conflict of interest.