Bio-PCR方法检测葫芦科作物种子携带西瓜嗜酸菌的特异性引物筛选

Screening of specific primers for detection of Acidovorax citrulli from cucurbits seed by Bio-PCR

由西瓜嗜酸菌(Acidovorax citrulli)引起的细菌性果斑病是一种毁灭性的种传病害,可为害多种葫芦科作物并造成重大经济损失。该病原菌为检疫性有害生物,种子带菌是田间病害发生的最重要初侵染来源,因此,种子健康检测成为病害综合防控过程中的重要环节。Bio-PCR是当前种子携带细菌检测的常用方法,而特异性引物的选择和使用是检测的关键。本研究使用已报道的7对引物对17株西瓜嗜酸菌、10株嗜酸菌属其它种的菌株和6株其它属的植物病原细菌进行了BioPCR检测,筛选出对西瓜嗜酸菌特异性最好的引物为SEQID4m/SEQID5。研究表明:使用该引物对西瓜嗜酸菌M H21纯菌菌悬液的检测限度为10~2CFU·m L^(-1);在人工添加菌悬液的模拟带菌西瓜种子中,使用ASCM和EBBA两种半选择性培养基结合引物SEQID4~m/SEQID5进行Bio-PCR检测,ASCM对种子中带菌量的检测限度可达到0.01 CFU·g^(-1),EBBA对种子中带菌量的检测限度为0.1 CFU·g^(-1)。

Bacterial fruit blotch （BFB）, caused by Acidovorax citrulli, is one of the most destructive seed-borne diseases of watermelon and other cucurbits. This seed-borne quarantine disease has been reported in most watermelon and melon producing regions worldwide. Since the infected seeds are the most important prima- ry inoculum source of BFB, seed health testing （SHT） and subsequent elimination of the contaminated seeds is one of the most effective methods to control the disease. Bio-PCR is Widely used in SHT for the detection of A. citruUi. As A. citrulli has abundant genetic diversity and many other closely related species, specific and sensitive primers are very important for the success of the seed health testing. To screen the best primers for detection of A. citrulli by Bio-PCR, 33 bacterial strains including 17 strains of A. citrulli, 10 strains of other Acidovorax spe- cies and 6 strains of other genera of plant pathogenic bacteria were chosen to evaluate 7 pairs of primers reported so far. Results indicated that the primer pair SEQID4m/SEQID5 had the best specificity that could distinguish all the tested strains of A. citrulli from those of other species and genera. The sensitivity of this primer pair was fur- ther tested with the Bio-PCR assay of A. citrulli. Results showed that the detection limit of the assay was 102 CFU · mL-1 for the pure bacterial culture, while for watermelon seeds the detection limit were 0.01 CFU ·g^-1 and0.1 CFU ·g-1 when seed extracts were cultured on semi-selective media ASCM and EBBA, respectively.