本實驗利用二組引子以聚合酶連鎖反應的方法,能同時檢出 ZmaR及Zwit這兩個基因,且能有效率的自本土蘇力菌株篩選出能產生 雙效菌素A的菌株,在25株本土蘇力菌的篩選中,找到一株具有最高抑 菌活性的菌株D1-10a,能抑制黃色微球菌(Micrococcus luteus BCRC 10449)、胡蘿蔔軟腐桿菌(Erwinia carotovora BCRC 12613)、蘭花鐮 胞菌(Fusarium solani)、甜椒疫病菌(Phytophthora capsici)、柑桔疫 病菌(Phytophthora citricola)、薑軟腐病菌(Pythium myriotylum)。在 培養時加入鐵離子能提高其抑菌活性。利用離子交換樹脂純化後的菌 液上清液,具抑制軟腐病菌菌絲生長能力。純化後產物利用高效液相 層析儀分析的結果,可在6.5到7.5分鐘間獲得一個單一的清楚高峰,此 物質的分子量約在387.1 Da,與仙人掌桿菌UW85所產生的雙效菌素A 類似,而這個雙效菌素A的類似物亦有抑制薑軟腐病菌之游走子發芽管 萌發的效果,本研究為台灣針對雙效菌素A的首次報告。
Using polymerase chain reaction (PCR) with two pairs of oligonucleotide primers to detect both ZmaR and Zwit genes. In this study, we were able to efficiently screen the local Bacillus thuringiensis (B.t) isolates that produced zwittermicin A. Of the 25 B.t isolates tested, B.t D1-10a exhibited the highest antifungal activity which can inhibit the growth of Micrococcus luteus、Fusarium solani、Phytophthora capsici、 Phytophthora Citricola、Pythium myriotylum.Antifungal activity can be enhanced by the addition of ferric ion to trypticase soy broth. And the cation-exchange hromatography (Amberliter IRC-50) purified supernatant of broth still showed inhibition against the mycelial growth (from zoospores) of Pythium myriotylum.The antibiotic substances were further purified by reverse phase C18 HPLC. A single peak was eluted at 6.5 to 7.5 minutes from HPLC. The antibiotic substances from HPLC were identified by LC-TOF MS. The molecular weight of this compound was determined to be approximately 387.1 Da, that was similar to the confirmed zwittermicin A producted by B.cereus UW85. In this study, the purified zwittermicin A-like compound can also inhibit the germination of P. myriotylum zoospores.