Citrus Plant Feasibility Study

Citrus aurantifolia plantlets were obtained from southern parts of Iran. Plants were kept in a humid greenhouse with 16:8 light: dark period. The plants were pruned to produce uniform new branches and leaves. NIGEB-88, an isolate of Xanthomonas citri subsp. citri (Xcc), was provided by the Bacterial Citrus Canker Collection of National Institute of Genetic Engineering and Biotechnology (NIGEB). The bacterium was cultured on YPGA (3 g/l yeast extract, 5 g/l peptone, 7 g/l glucose solidified with 15 g/l agar) at 28 °C for 48 h. A single colony was subcultured in YP medium (3 g/l yeast extract and 5 g/l peptone) on a rotary shaker at 180 rpm at 28 °C till OD600 = 0.3, i.e., 5 × 108 cfu/ml. To prepare the inoculum, the culture was centrifuged for 10 min at 5000 ×g and the pellet was suspended with dH2O to reach a concentration of 1 × 105 cfu/ml. For inoculation, host plant leaves were surface-sterilized with ethanol and the inoculum (500 µl of 1 × 105 cfu/ml) was injected into the parenchymal space on the abaxial leaf side. Control leaves were treated similarly

Spot detection, gel matching and interclass analyses were performed with Melanie 6.0 software (Gene Bio, Geneva, Switzerland). Default detection parameters were used for spot detection and spot pairing controlled manually. Relative mass (Mr) and isoelectric point (pI) of spots were calculated by standard protein markers. Normalized percent volumes of spots based on the total intensity of valid spots were calculated and used for statistical quantitative analysis. Only spots that were discovered on all three replicate gels were selected for further analyses. Univariate statistical analyses were carried out by SPSS (Version15, SPSS Inc, USA) software. Spots with statistically significant difference (P < 0.05) between the infected vs. control plants were considered as candidates for probable roles in defense