Mutation of Bacillus licheniformis using low-energy ion beam bombardment

Anthracnose, caused by the fungus Colletotrichum sp., is an important disease affecting a wide range of flowers. To control this fungus various methods including the use of natural antagonists have been used for biological control. In this study, Bacillus licheniformis, isolated from hot springs in the Chiang Mai province, was shown to suppress conidia germination of the fungus and reduce symptoms caused by the disease in planta. To induce mutations in B. licheniformis, a low-energy ion beam was applied to the bacteria using N-ions to bombard the bacteria under vacuum conditions at an energy of 28-50 keV with a fluence range of 1-10 × 1015 ions/cm2. After this treatment, one mutant was found which had lost its antagonistic property. To try to characterize this mutation, the HAT-RAPD technique was used to produce band patterns for both the bombarded bacteria and the original wild type. From these polymorphic band patterns, differences were found which were induced by the ion beam. These polymorphism bands were subcloned into a pGEM-T vector and sequenced. One-band fragment conserved in the wild type and lost in the mutant bacteria was found to code for the lipase gene. Further research will focus on other genes and gene functions which may be involved in the observed antipathogenicity.