巢式PCR检测琯溪蜜柚黑斑病病原菌

Detection of Pathogens Causing Citrus Black Spot of Guanxi Honey Pomelo by Nested PCR

利用特异性引物从琯溪蜜柚黑斑病菌基因组DNA中扩增出一条分子量为487 bp的特异性条带,准确地与疮痂病、炭疽病和黄斑病菌等蜜柚常发性真菌病害区分开。采用巢式PCR对该引物的检测灵敏度进行测定,结果显示,对于黑斑病菌基因组DNA,巢式PCR的检测灵敏度为100 fg/μL,灵敏度比常规PCR至少提高100倍;对于发病组织,巢式PCR可从病斑组织含量为100μg的DNA样品中检测到黑斑病菌。采用巢式PCR检测技术,可从蜜柚的黑斑病显症组织和未显症组织特异性地检测到病原菌,且检出率分别为96.67%和90%。

Citrus black spot is a fungal disease in most Guanxi honey pomelo. By using the specific primers, a unique 487 bp band was obtained by PCR amplification from genomic DNA of the pathogen causing citrus black spot of Guanxi honey pomelo and the method could accurately distinguish the honey pomelo black spot pathogen from the other disease pathogens of Sphaceloma fawcetti Jenk, Colletotrichum gleosporioides Penz and Mycosphaerella cirri White-Side in Guanxi honey pomelo. The sensitivity was determined by nested PCR. For the genomic DNA of honey pomelo black spot pathogen, the sensibility of nested PCR reached 100 fg/p^L, 100 times higher than that of routine PCR. For the DNA of diseased tissues, the sensibility of nested PCR reached 100 Ixg of DNA sample. Through nested PCR assay, the pathogen of honey pomelo black spot could be specifically detected form diseased and symptom latent infection honey pomelo tissues with the detection rate of 96.67% and 90%, respectively.