KOWIBUCHA FILTRATE AS AN INHIBITORY FACTOR FOR CUCUMBER MOSAIC CUCUMOVIRUS AND BACTERIAL CONTAMINATION IN BANANA TISSUE CULTURES

摘要

Great problems facing banana crop production and tissue culture are virus infection and bacterial contamination. Banana suckers, cultivar Maghrabi (50 cm height) confirmed to be infected with Cucumber mosaic cucumovirus (CMV) via indirect enzyme linked immunosorbant assay (I-ELISA) and specific polyclonal antiserum were used for such investigation. As a control total bacterial plate count and CMV I-ELISA values were determined for ten untreated banana shoot tips (1 cm in length). Another ten shoot tips of the same banana variety, age, length and growing conditions were determined once more, after disinfection with 4% sodium hypochlorite or with undiluted kombucha (growing with or without adding 0.2% folic acid) filtrate obtained using 0.45 um Millipore~R filter for removing microbes. Data proved that sodium hypochlorite was more effective as a disinfectant, while kombucha filtrate slightly reduced virus I-ELISA values. After disinfection with sodium hypochlorite, banana meristem tips (0.5 cm in length) were cultured on Murashige and Skoog medium (MS) supplemented with 3 mg BA, 3% sucrose and integrated with 1.0 ml kombucha (growing with or without adding 0.2% folic acid) filtrate per 15 ml medium. At time intervals of one and three month banana tissues were studied for bacterial count and CMV I-ELISA values. Best results were obtained with kombucha growing with 0.2% folic acid as an enhancer, as its addition prevented bacterial contamination by 50-60% after one and three months, respectively. Onthe other hand it decreased CMV I-ELISA values and produced a reasonable number (60%) of virusfree bananas. The contaminating predominant bacteria were identified to be related to Bacillus cereus depending on morphology and biochemical tests. Kombucha filtrate protein content was determined spectrophotometry and by gel electrophoresis. CMV was detected and identified in bananas using immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) for the coat protein gene (cp) and found to berelated to CMV subgroup I.