Hansen BM, Damgaard PH, Eilenberg J, Pedersen JC
Department of Marine Ecology and Microbiology, National Environmental Research Institute, Frederiksborgvej 399, Roskilde, DK-4000, Denmark.
J Invertebr Pathol. 1998 Mar;71(2):106-14. doi: 10.1006/jipa.1997.4712.
Bacillus thuringiensis isolates from the phylloplane of organically cultivated cabbage were characterized using molecular and phenotypic methods. Of the 58 isolates under study, 31 belonged to serovar kurstaki, 16 did not react with any of the currently recognized antisera, 7 reacted with known antisera, and 4 could not be serotyped as they were nonmotile. Round crystals were found in 26 isolates, while bipyramidal crystals were found in the remaining 32 isolates, all of which had activity to lepidopteran larvae. Further, one isolate with unknown serotype and round crystals had lepidopteran activity. Colony hybridization was found to be a useful tool for screening the isolates for specific gene homologies and showed good correlation with the phenotypic observations. Polymerase chain reaction (PCR) was used for confirmation of the colony hybridization data, in most cases with concordant results. However, in one case some of the colony hybridization data could not be confirmed by PCR, due to DNA sequence variations in the binding area of one of the primers. The random amplified polymorphic DNA (RAPD) analysis showed that isolates otherwise indistinguishable could be distinguished by this method. However, the method was not able to distinguish the 31 kurstaki isolates. Further, the kurstaki isolates could not be distinguished from the B. thuringiensis serovar kurstaki HD-1 strain used in commercial products for lepidopteran control. One of the isolates was a serovar israelensis, but no genes encoding dipteran activity could be detected, and the RAPD analysis revealed that the DNA fingerprint of this israelensis isolate deviated from the israelensis ONR60A isolate used in commercial products. In conclusion we find that a molecular method like colony hybridization is suitable for screening large collections of bacteria. When colony hybridization data are combined with RAPD analyses isolates can be grouped based on genetic potential and DNA fingerprint, whereby further characterizations by PCR and the more labourious phenotypic methods can be performed more effectively. Copyright 1998 Academic Press.
采用分子和表型方法对有机种植甘蓝叶面上的苏云金芽孢杆菌分离株进行了鉴定。在所研究的58株分离株中，31株属于库尔斯塔克血清型，16株不与任何目前已知的抗血清发生反应，7株与已知抗血清发生反应，4株因无运动性而无法进行血清分型。在26株分离株中发现了圆形晶体，其余32株分离株中发现了双锥形晶体，所有这些分离株对鳞翅目幼虫均有活性。此外，一株血清型未知且有圆形晶体的分离株具有鳞翅目活性。发现菌落杂交是筛选分离株特定基因同源性的有用工具，并且与表型观察结果具有良好的相关性。聚合酶链反应（PCR）用于确认菌落杂交数据，在大多数情况下结果一致。然而，在一个案例中，由于其中一个引物结合区域的DNA序列变异，一些菌落杂交数据无法通过PCR得到确认。随机扩增多态性DNA（RAPD）分析表明，该方法可以区分原本难以区分的分离株。然而，该方法无法区分31株库尔斯塔克分离株。此外，库尔斯塔克分离株与用于商业鳞翅目防治产品中的苏云金芽孢杆菌库尔斯塔克HD-1菌株无法区分。其中一株分离株是以色列血清型，但未检测到编码双翅目活性的基因，RAPD分析表明该以色列分离株的DNA指纹与商业产品中使用的以色列ONR60A分离株不同。总之，我们发现像菌落杂交这样的分子方法适用于筛选大量细菌。当菌落杂交数据与RAPD分析相结合时，可以根据遗传潜力和DNA指纹对分离株进行分组，从而可以更有效地通过PCR和更繁琐的表型方法进行进一步鉴定。版权所有1998年学术出版社。
