烟草内生芽孢杆菌降低烟叶亚硝胺类物质含量的研究

Reduction of tobacco specific nitrosamine in air-cured tobacco leaves by a bacterial endophyte(Bacillus sp.)

从白肋烟TN90品种的主脉组织中分离到1株内生菌株WT。根据形态和生理生化特征进行鉴定。该菌株在TSA培养基上菌落形态为白色，环状突起。对利福平和萘啶酮酸敏感。革兰氏阳性（G^＋），催化酶活性，产生芽孢。可运动，杆状。具一定的碱性磷酸酶活性和一定的脲酶活性，鉴定为芽孢杆菌（Bacillus sp．）。在温室移栽时将该内生细菌接种于白肋烟TN90。并在收获后喷施细菌悬液于烟叶表面，进行亚硝胺类物质的检测，结果表明：接种WT可减少白肋烟TN90的亚硝胺组分，TSNA总量比对照减少，叶片组织减少21．7％～44．6％，主脉组织减少16．7％-80％。整个根系接种可有效降低叶片和主脉组织内烟草特有亚硝胺（TSNA）总量（P≤0．05），分别降低了38．0％和80％。叶面喷施可使叶片组织内烟草特有亚硝胺（TSNA）总量降低44．6％，也明显影响叶片和主脉组织中N-亚硝基去甲基烟碱（NNN）含量，差异显著（P≤0．05）。对N-亚硝基新烟草烟碱（NAT）和N-亚硝基假木贼碱（NAB）含量的影响不显著。

A putative tobacco bacterial endophyte, WT was isolated from Burley tobacco variety TN90,and it was characterized by morphological,physiological and biochemical methods. The WT isolate formed white, convex raised colonies on tryptic soy agar medium that were extremely sensitive to rifampicin and nalidixic acid. It was Gram positive, catslase positive, spore-forming, motile rod,with limited alkaline phosphatase activity and limited nitrate-reducing ability. Identification of WT by substrate use patterns（ BIOLOGTM ） was unsuccessful but the most likely genus in which to place the isolate is Bacillus sp.. TN90 was grown in a greenhouse and at the transplant stage inoculated with isolate WT,which was unable to reduce NO3 ^- to NO2^ -. The grown tobacco was air cured and an additional treatment in which WT was sprayed on leaf tissue was employed. The total nitresamine content and the content of individual nitrosamlnes was, on average,lower in inoculated plants than in the control. Total nitrosamine content reduced 21.7% to 44.6% in lamina tissue and 16.7% to 80% in midrib tissue. In three inoculation methods,wholeroot inoculation could effectively reduce nitremine content with the percentage 38.0% in lamina tissue and 80% in midrib tissue（ P≤0.05 ）. And Sprayed plants were as effective at reducing nitresamine content in lamina tissue as plants inoculated at transplanting with the percentage of 44.6%, and effectively reduced NNN content in lamina and midrib tissues （ P ≤0.05 ）, however, the reduction in NAT and NAB content was not statistically significant.