| Tobacco black root rot disease,caused by Thielaviopsis basicola, is destructive to tobacco production throughout the world. T. basicola, a soil-borne pathogen, is widely distributed in areas of intensive tobacco cultivation in the world and it is hard to control. The diseased residual and soil with the pathogen become the primary source of infection of tobacco black root rot disease. Once the condition is appropriate, the conidia or chlamydospores germinate to form invasive hypha and penetrate the host epidermal cells. After invasion, the hypha form a spread of branches between the epidermal cells, produce a large number of conidia and chlamydospores which can infect hosts again. Without effective fungicides or resistant varieties to control the disease, it is urgent to develop early diagnosis to detect and quantify T. basicola quickly and exactly in its latent form in soil in order to prevent the development of the tobacco black root rot. The traditional detection methods have many disadvantages such as long detecting cycle, complicated operation and low sensitivity, which can not meet the requirement of the rapid disease-detection. With the development of molecular biological technology, Polymerase Chain Reaction (PCR) has been widely used for the detection of pathogenic microorganism because of its rapidity, specificity and sensitivity. With the PCR technology in this research, we successfully developed the conventional PCR and qPCR assays for quantitative analysis of T. basicola in soil.In this study, a primer pair Tb5/Tb6 targeting ITS regions of rDNA of T. basicola was successfully developed, which gave a specific amplicon of 338 bp with T. basicola DNA. The conventional PCR assay was specific and sensitive and the detecting sensitivity was 1 pg/μL for the genome DNA of T. basicola and 10 conidia per reaction for the artificially infested soil. The result showed that the assay can be applied to detecting T. basicola in soil.The SYBR Green? I PCR detection assay for quantification of T. basicola in soil was developed based on the conventional PCR described above. Similarly, the primer pair Tb5/Tb6 was used to establish the SYBR Green? I PCR assay. In SYBR Green? I PCR, with a series of 10-fold DNA dilutions of T. basicola, the detection limit of 100 fgμL-1 of genomic DNA was achieved. With DNA from the soil inoculated with different number of T. basicola conidia,the detection limit was 0.4 conidia per reaction in artificially infested soil, and the correlation coefficient between the Log number of pathogen conidia and the PCR cycle values was 0.95(P<0.05). Compared to conventional PCR assay, SYBR Green? I PCR assay was 10 times more sensitive for the genomic DNA and 25 times more sensitive for the soil DNA.The TaqMan PCR detection assay for quantification of T. basicola in soil was also developed. A primer pair and TaqMan probe were designed using the program of Beacon designer to develop the TaqMan probe detection assay, which gave a specific amplicon of 76 bp with T. basicola DNA. With a series of 10-fold DNA dilutions of T. basicola, the detection limit was 100 fgμL-1 of genomic DNA. With DNA from the soil inoculated with different number of T. basicola conidia, the detection limit was 0.3 conidia per reaction in artificially infested soil, and the correlation coefficient between the Log number of pathogen conidia and PCR cycle values was 0.98(P<0.05).Compared to the conventional PCR assay, TaqMan PCR assay was 10 times more sensitive for the genomic DNA and 33 times more sensitive for the soil DNA. The conventional PCR and qPCR assays established in this study could be applied to detecte and quantify T. basicola in soil accurately, and it is valuable for dynamic monitoring of the occurrence of the disease in the field. As a result, the disease can be diagnosed early as well as the detection system can provide a theoretical basis for the disease prevention and control.
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