Development of Standard Inoculation Procedures for Blood Disease Bacterium on Banana

Tee, Shuan Ling (2018) Development of Standard Inoculation Procedures for Blood Disease Bacterium on Banana. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

The Ralstonia solanacearum is a bacterial pathogen with wide host range that causes bacterial wilt particularly in solanaceous plant and banana. This bacterium has caused a serious disease in banana called blood disease and has severed the banana industry in Malaysia, earning the nickname Blood Disease Bacterium (BDB). This research was undertaken to understand the growth of BDB under the in-vitro condition and develop a standard growth curve specifically for BDB. The BDB’s growth curve developed was further utilised to develop a standard inoculation procedure for this disease. The BDB bacterium was isolated from infected banana plant sampled from Melaka. The Kelman’s tetrazolium chloride (TZC) medium was used to isolate and purify the BDB from plant samples. Pure culture of the BDB was then multiplied using nutrient agar (NA) for growth curve development and inoculation purposes. Colony forming unit (CFU) count was performed for BDB with various dilutions prepared and measured using spectrophotometer which is the percent transmittance with the setting of 600nm wavelength. The CFU values counted were then used for development of growth curve. Standard curve developed was then utilised for the preparation of BDB suspensions with concentration of 106,108, 1010 and 1012 CFU/ml for inoculation purpose. Banana seedlings (Kapuk variety) with 4-5 leaf stage were chosen for the inoculation due to their susceptibility to BDB. Each BDB treatment was repeated three times with sterile distilled water used as control treatment. Disease incidence and severity were observed and recorded post inoculation on weekly basis. This experiment has successfully induced the BDB symptoms on all BDB inoculated seedlings with 100% disease incidence. The disease severity recorded has showed that the BDB concentration of 106 CFU/ml is sufficient to cause the BDB symptom. Successful in re-isolation of the BDB from the wilted seedlings have fulfilled the Koch’s postulates. The calibrated methods of plant pathogen inoculation have proven their feasibility which could contribute to better accuracy plant pathogen screening and development of resistant plant variety.