Citrus Ivarina Case Study

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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 with dH2O. Mock-inoculated and infected plants were sampled 1, 4 and 7 days post-treatment. The sampling process occurred biologically thrice, all of which were immediately frozen in liquid nitrogen and kept finally at - 80C for further analyses. 2.2. Extraction of the apoplastic fluid Leaf apoplastic fluid were obtained from the whole citrus leaves by vacuum-infiltration-centrifugation method (Dani et al., 2005). C.

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