Direct Detection of Race 2 Ralstonia solanacearum (Smith) Yabuuchi in Banana Fruit Stalk Using Polymerase Chain Reaction

摘要

A direct method for the detection of Ralstonia solanacearum in bugtok/moko- and bacterial wilt-infected banana fruit stalk by polymerase chain reaction (PCR) using the specific Rs banana strain 24F/24R primers was developed. R. solanacearum was initially not detected using the DNA directly isolated from banana fruit stalk and from ooze suspension using the modified Porebski CTAB-based method, UltraClean~(TM) Soil DNA Kit, MasterPure~(TM) Plant Leaf DNA Purification Kit and PrepMan~(TM) Ultra Sample Preparation Reagent. However, enrichment by plating the ooze suspension on Kelman's Agar (KA) and incubation for 15 h allowed the detection of the pathogen at 10~8 cfu g~(-1) sample using the above techniques. Also, just boiling the resulting growth in water for 10 min and using the boiled extract for template in PCR allowed the detection of the pathogen. Directly plating the infected tissue on KA and boiling the resulting growth also gave a positive result in PCR. This method was tried in naturally-infected samples, and there was positive detection of the organism in 73% of putative moko-and bugtok-infected fruit stalk samples. The negative results from the rest of the samples may be due to misdiagnosis of the disease, as the basis was only on morphological signs. This method was very useful in showing that not all fluidal colonies with reddish centers obtained from isolation plates are R. solanacearum as reported in the literature. Moreover, directly boiling the ooze suspension for 10 min from naturally-infected samples without enrichment also produced positive results at the same level of sensitivity. This method can be utilized for diagnosis/confirmation and monitoring in R. solanacearum-mfected samples. As far as we know, this is the first PCR-based direct detection method developed for R. solanacearum from fruit stalks.