检疫性真菌病害向日葵黑茎病菌和茎溃疡病菌的三重PCR分子检测

Triplex-PCR Molecular Detection of Two Quarantine Fungal Diseases of Sunflower,Leptosphaeria lindguistii and Diaporthe helianthi

向日葵黑茎病菌Leptosphaeria lindguistii和向日葵茎溃疡病菌Diaporthe helianthi是向日葵上寄生的2种重要的检疫性真菌病害.针对这2种致病菌,目前国内外已有形态学鉴定和危害的研究报道以及向日葵黑茎病菌的ITS序列分析和RFLP初步研究,但未见有2种病原菌同步分子检测的报道.本研究首先通过培养,观察比较了2种病原菌的菌落和形态特征.发现前者菌落絮状,分生孢子器深褐球型,分生孢子为单胞肾形;后者菌落短绒毛状,分生孢子器柠檬黄,分生孢子为线型.通过油菜黑茎病菌L.biglobosa的肌动蛋白基因设计供试材料的通用引物act F/act R,扩增产物片段约为720bp,然后采用真菌通用引物ITS1/ITS4和引物act F/act R,针对所有菌株菌丝DNA进行ITS区域和Actin基因PCR扩增,经克隆测序结果比对分析发现,向日葵黑茎病菌和向日葵茎溃疡病菌的ITS区域存在极高的相似度.因此,依据Actin基因特异位点分别设计出向日葵黑茎病菌和向日葵茎溃疡病菌的特异引物LLF/LLR和DHF/DHR.在同一PCR管中3组引物(ITS1/ITS4,LLF/LLR和DHF/DHR)经优化体系后,ITS1/ITS4扩增序列作为所有菌株菌丝基因组DNA提取模板的质量监控,针对向日葵黑茎病菌和向日葵茎溃疡病菌单一模板的ITS区域与Actin基因中的两个位点同时进行三重PCR扩增.利用此方法检测向日葵黑茎病菌出现580bp的ITS保守区扩增片段和255bp的Actin基因的特异扩增带,向日葵茎溃疡病菌出现580bp的ITS保守区扩增片段和394bp的Actin基因特异扩增带.所有菌株均未见非特异性片段的干扰.此三重PCR检测体系的建立为此两种检疫性真菌病害的同步分子检测提供了特异方法.

Leptosphaeria lindguistii and Diaporthe helianthi are two important quarantine fungal diseases of sunflower. The morphological observation of the two pathogens and the degree of their damage to sunflower have been reported; the PCR product sequencing of ITS region and RFLP analysis of sunflower black stem disease （L. lindguistii） have also been reported at home and abroad. But so far no simultaneous molecular detection of the two pathogens has been reported. In this study, the colony and morphological characteristics of L. lindguistii and D. helianthi were observed and compared. The colony of the former was flocculent, its pycnidia were dark brown balls in shape, and its conidial cells were kidney-shaped, while the colony of the latter was velutinous, its pycnidia were lemon yellow balls in shape, and its conidia cells were in linear form. Based on the aetin gene sequence of L. biglobosa, the universal primer act F/act R was designed and used to amplify the mycelial DNAs of the experimental materials. Uniformly a frag- ment of 720bp approximately was obtained; it was discovered that the ITS region and actin gene of all mycelial DNAs amplified by primers of ITS 1/ITS 4 and act F/act R had high similarity in the ITS region of L. lindguistii and D. helianthi; the specific primer pairs, LLF/LLR and DHF/DHR, for L. lindguisti and D. helianthi, respectively, were designed, based on their difference in the actin gene. Three pairs of primers, ITS 1/ITS 4, LLF/LLR and DHF/DHR, were used simultaneously for triplex PCR amplification of all the myeelial DNAs after system optimization, with the ITS1/ITS4 amplified sequence as quality control of the extracting genomic DNA template. For L. lindguistii, triplex PCR detected a 580 bp fragment of ITS conserved region and a 255 bp specific band of the actin gene; for D. helianthi, triplex PCR detected a 580bp fragment of ITS conserved region and a 384bp specific band of the actin gene. No nonspecific bands appeared on triplex PCR of all the tested strains. Thus, the established triplex PCR detection is a specific and operative method to simultaneously detect the two quarantine fungal diseases, L. lindguistii and D. helianthi, of sunflower.