Using Real-time PCR Measurements For Detection Of Carbendazim Resistance In Sclerotinia Sclerotiorum

Rape sclerotiniose is the main rape disease in China, influencing greatly the production of rape. At present, the most popular fungicide used to control S. sclerotiorum is benzimidazol, especially carbendazim. Benzimidazole fungicides bind to β -tubulin, inhibiting microtubulin function or disassembling microtubulin, further prevent cell's mitosis of fungi for growth. Because benzimidazoles are characterized by high specificy, single action position, and high application frequency, many plant pathogenic fungi occurred resistance to these fungicides after 2-3 years' application. The carbendazim resistance in S. sclerotiorum was found to be correlative to a single point mutation resulting in the replacement of glutamic acid 198 by alanine in the gene coding P-tublin protein.To detect and monitor the resistance frequcncy of S.sclerotiorum in field quickly and accurately, two real-time PCR methods were designed according to the single point mutation of MBC^HRisolates. One method was designed on the basis of a principle that two TaqMan probes labeled different reporter respectively on its ends can be linked with templates respectively to give out fluorescent; the an other method combined SYBR GREEN I fluorescent dye with ASO-PCR detection technology to detect the resistant strains as well as total sample population quantitatively.In order to determine the resistance frequency of a fungus population in a single reaction tube, a pair of universal primers TYF and TYR , a TaqMan probe for sensitive strains ( 5' end labeled reporter Rox, and 3' end labeled quencher Tarma) and a probe for resistant strains (5' end labeled reporter Fam, and 3' end labeled quencher Tarma) were developed. In the experiment, the reaction condition is optimized for times, but the detection couldn't be realized, and the specificity of resistant probe was low so that couldn't combine with resistant template specificially.In the method with SYBR GREEN I dye, a pair of primers RFP4 and RRP were designed to amplify the β-tubulin gene in the resistant S.sclerotiorum according to the principle of ASO-PCR, and the 198-th mutative coder is used as its β-end base. In addition, according to the difference of the intron of P-tubulin gene of S.sclerotiorum with othercommon filaceous fungi, a pair of specific primers SclSF and SclAF were designed to amplify the specific fragment of S.sclerotiorum. The use of two pairs of primers ensured the reliability and veracity of the detection. By means of SYBR GREEN I dye, the sensitivity of the detection greatly increased, with the minimum detection of 0.0028ng template DNA, which is above ten bold than common ASO-PCR. On the other hand, total DNA can be extracted directly from sclerotinia, instead of dealing with each strain singly, so the time and workload is greatly deceased. It takes only 4 hours in this method, from the extraction of genome DNA to the accomplish of real-time PCR detection.In the study, the quantitative standard curve of the detection of carbendazim-resistance S.sclerotiorum was established through real-time PCR with SYBR GREEN I dye. The calibration equation of the resistant strain is Y=-3.45X+25.60 (R2=0.998), and the calibration equation for the quantification of the total S.sclerotiorum is Y=-3.41X+27.30 (R2=0.999), in which Y represents Ct; X represents logarithm of mass of template. On the basis of above equation, 200 S.sclerotiorum strains ,collected from fields of 4 towns in TongZhou, Jiangsu province, were detected. The result showed that (36.83 ±0.09)% of total strains have carbendazim resistance, which coincides with the result of mycelial growth tests ( 35.9% of total 223 strains) and common ASO-PCR method (39% of total 100 strains).