十字花科作物的黑腐病是由油菜黄单胞菌引起的。卷心菜(Xcc),大大降低了农作物的生产力。三场Xcc比赛,比如1、4和6种族,已经从印度发现了9个avr基因,或III型效应子(T3Es)。这里,我们使用了三个T3Es-avrXccC,avrBs1和avrGf1从细菌DNA中鉴定Xcc,细菌悬浮液,Xcc感染的种子,和感染叶片的汁液使用多重PCR。使用具有Xcc的gDNA的基因特异性引物扩增T3Es。然后,对多重PCR进行了优化,并使用黑腐病感染的花椰菜叶的汁液扩增了T3Es。Further,这种方法从人工感染的花椰菜种子(1-100%)和在营养琼脂培养基上生长的Xcc菌落(0.1-100%)中扩增了T3Es。T3E基因的引物特异性表明,这些基因在所有印度Xcc菌株和种族中都被特异性检测到,虽然没有观察到其他无关细菌的条带,如X.euvesicatoria,X.稻米pv.稻米,荧光假单胞菌,青枯拉尔松,枯草芽孢杆菌,和解淀粉芽孢杆菌。Further,这种PCR具有很高的灵敏度,并使用高达0.01ngXccDNA扩增T3E基因。基于T3Es的多重PCR的高特异性和敏感性使其成为一种潜在的方法,可用于从各种模板扩增Xcc,如纯化的DNA,Xcc感染的种子和叶子,粗提物,等。,无需提取植物或细菌DNA。
■在线版本包含补充材料,可在10.1007/s13205-023-03691-z获得。
The black rot disease in crucifer crops is caused by Xanthomonas campestris pv. campestris (Xcc) which drastically reduces the productivity of crops. Three Xcc races, such as races 1, 4, and 6, have been identified from India that possess nine avr genes, or type-III effectors (T3Es). Here, we used three T3Es-avrXccC, avrBs1, and avrGf1 to identify Xcc from bacterial DNA, bacterial suspensions, Xcc-infected seeds, and the sap of the infected leaves using multiplex PCR. The T3Es were amplified using gene-specific primers with gDNA of Xcc. Then, the multiplex PCR was optimized and amplified T3Es using the sap of black rot-infected cauliflower leaves. Further, this method amplified T3Es from artificially infected seeds (1-100%) of cauliflower and from Xcc colonies (0.1-100%) grown on nutrient agar medium. The primer specificity of T3E genes elucidates that these are specifically detected in all Indian Xcc strains and races, while no bands were observed with other unrelated bacteria, such as X. euvesicatoria, X. oryzae pv. oryzae, Pseudomonas fluorescens, Ralstonia solanacearum, Bacillus subtilis, and B. amyloliquefaciens. Further, this PCR possesses high sensitivity and amplifies T3E genes using up to 0.01 ng Xcc DNA. The high specificity and sensitivity of T3Es-based multiplex PCR make it a potential method and can be used to amplify Xcc from various templates, such as purified DNA, Xcc-infected seeds and leaves, crude extracts, etc., without the need to extract plant or bacterial DNA.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s13205-023-03691-z.