Nested PCR assay for detection of Corynespora leaf fall disease caused by Corynespora cassiicola
Nested PCR assay for detection of Corynespora leaf fall disease caused by Corynespora cassiicola
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巢式 PCR 检测由 Corynespora cassiicola 引起的棒孢落叶病
DOI：
10.1071/ap08086
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发表时间：
2009
影响因子：
1.4
通讯作者：
H. Zhang
中科院分区：
文献类型：
--
作者：
Y. Qi;X. Zhang;J. Pu;Y. Xie;H. Zhang;S. L. Huang;S. Li;H. Zhang
由 Corynespora cassiicola 引起的巴西橡胶棒棒孢落叶病已成为亚洲和非洲橡胶胶乳产量严重减少的主要原因。本研究的目的是确定中国海南省和云南省是否存在 C. cassiicola 生理小种，并加速和简化诊断过程。我们对 4 个不同橡胶无性系（基因型 PR107、大丰 95、RRIM600 和热研 7-33-97）的离体叶子上的 17 个 C. cassiicola 分离株进行了致病性测试。致病性测试显示，其中 16 个分离株对 RRIM 600 具有致病性，这证实这些分离株为 C. cassiicola 小种 1。使用 C. cassiicola 物种特异性引物组（CCF 和 CCR-2）进行初始 PCR 来扩增 ITS1-5.8S-ITS2 区域的一部分。长度为 272 bp 的 DNA 片段表明 C. cassiicola 的存在。为了提高灵敏度，开发了巢式PCR测定法，使用来自初始PCR的稀释的(1:100)扩增产物作为模板以及物种特异性寡核苷酸CCF和CCR-1作为引物来获得预期长度(152bp)的DNA片段。巢式PCR可检测到的真菌DNA的最低浓度为168 fg，而初始PCR中的浓度为16.8 pg。因此，使用巢式PCR将检测灵敏度提高了100倍。此外，在接种后3天，在人工感染的橡胶树中可以检测到病原体。由于其高灵敏度、特异性和可靠性，巢式 PCR 可用于筛选和认证年轻的不含 C. cassiicola 的橡胶树，以便分发给商业种植者。
Corynespora leaf fall disease of Hevea brasiliensis caused by Corynespora cassiicola has been a major cause of severely reduced rubber latex production in Asia and Africa. The aim of this study was to determine whether physiological races of C. cassiicola exist in the Hainan and Yunnan provinces, China, and to accelerate and simplify the process of diagnosis. We conducted pathogenicity tests with 17 C. cassiicola isolates on detached leaves of four different Hevea rubber clones (genotypesPR107, Dafeng 95,RRIM600 and Reyan 7-33-97). Pathogenicity tests showed that 16 of the isolates were pathogenic to RRIM 600, which confirms these isolates as C. cassiicola race 1. An initial PCR was performed to amplify a portion of the ITS1-5.8S-ITS2 region using a C. cassiicola species-specific primer set (CCF and CCR-2).DNA fragments of 272 bp in length indicated the presence of C. cassiicola. To improve the sensitivity, a nested PCR assay was developed to obtain DNA fragments of the expected length (152 bp) using diluted (1 : 100) amplified product from the initial PCR as the template and species-specific oligonucleotides CCF and CCR-1 as primers. The lowest concentration of fungal DNA that could be detected by nested PCR was 168 fg, while its concentration in the initial PCR was 16.8 pg. Therefore, the sensitivity of detection was enhanced 100-fold using nested PCR. In addition, the pathogen could be detected from artificially infected Hevea rubber trees 3 days after inoculation. Due to its high sensitivity, specificity, and reliability, nested PCR is useful for the screening and certification of young C. cassiicola-free Hevea rubber plants for distribution to commercial growers.