杧果露水斑病菌和细菌性黑斑病菌双重PCR体系的建立

Establishment of Double PCR System of Cladosporium cladosporioides and Xanthomonas campestris pv. mangiferaeindicae of Mango

杧果露水斑病和细菌性黑斑病在国内杧果产区普遍发生,严重威胁杧果安全生产,俗称杧果"两病"。建立准确高效的检测方法,提早检出杧果两病并及时采取防治措施,可有效减轻为害。本实验通过筛选反应体系和电泳条件,建立了可同时检测杧果中的露水斑病菌和细菌性黑斑病菌的双重PCR检测体系。结果表明:25μL反应体系,2×Taq PCR MasterMix 12.5μL,引物ML-SF9/SR5 (10μmol/L)各1.5μL、XcmHF/HR(10μmol/L)各1μL,模板各1μL,加ddH2O补足至25μL。反应条件为94℃预变性4 min;94℃变性45 s,65℃退火45 s,72℃延伸1 min,35个循环;72℃延伸5 min。电泳检测条件为1.5%琼脂糖凝胶浓度、120 V电压电泳25 min。该体系检测下限为杧果露水斑病原菌g DNA 0.224 ng/μL、细菌性黑斑菌液2.6×1~04cfu/m L。该技术可应用于田间杧果"两病"的早期检测与鉴定工作中。

Cladosporium cladosporioides(Fr.) de Vries and Xanthomonas campestris pv. mangiferaeindicae of mango called the "two mango diseases" occur in most mango producing regions of China, which seriously affects mango production. In order to effectively reduce the loss, it is necessary to establish an accurate and efficient detection method to detect two mango diseases and prevent them in time. By screening the reaction system and electrophoresis conditions in this study, a double PCR detection system for detecting Cladosporium cladosporioides(Fr.) de Vries and Xanthomonas campestris pv. mangiferaeindicae of mango was established. The results showed that 25 μL reaction system consisted of 12.5 μL 2×Taq PCR MasterMix, 1.5 μL 10 μmol/L ML-SF9, 1.5 μL 10 μmol/L ML-SR5,1 μL 10 μmol/L XcmHF, 1 μL 10 μmol/L Xcm HR, and 1 μL of each template, made up to 25 μL with ddH2 O.The reaction conditions were as follows: one cycle for pre-degeneration at 94℃ for 4 min, 35 cycles at 94℃ for 45 s for degeneration, 65℃ for 45 s for annealing, and 72℃ for 1 min for extension, followed by one cycle at 72℃ for 5 min for extension. The electrophoresis detection conditions were 1.5% agarose gel and electrophoresis under 120 V for25 minutes. The results showed that the lower limit was 0.224 ng/μL for Cl. cladosporioides g DNA and 2.6 ×10~4cfu/mL for Xcm using this double PCR detection system. This method could be applied to the early detection and identification of "two diseases" on mangoes in field.