Lin Zhenyue, Xu Shiqiang, Que Youxiong, Wang Jihua, Comstock Jack C, Wei Jinjin, McCord Per H, Chen Baoshan, Chen Rukai, Zhang Muqing
Guangxi University, Nanning, Guangxi, China; Fujian Agriculture and Forestry University, Fuzhou, China.
Guangxi University, Nanning, Guangxi, China.
PLoS One. 2014 Aug 20;9(8):e104195. doi: 10.1371/journal.pone.0104195. eCollection 2014.
Pokkah boeng disease caused by the Fusarium species complex results in significant yield losses in sugarcane. Thus, the rapid and accurate detection and identification of the pathogen is urgently required to manage and prevent the spreading of sugarcane pokkah boeng.
A total of 101 isolates were recovered from the pokkah boeng samples collected from five major sugarcane production areas in China throughout 2012 and 2013. The causal pathogen was identified by morphological observation, pathogenicity test, and phylogenetic analysis based on the fungus-conserved rDNA-ITS. Species-specific TaqMan real-time PCR and conventional PCR methods were developed for rapid and accurate detection of the causal agent of sugarcane pokkah boeng. The specificity and sensitivity of PCR assay were also evaluated on a total of 84 isolates of Fusarium from China and several isolates from other fungal pathogens of Sporisorium scitamineum and Phoma sp. and sugarcane endophyte of Acremonium sp.
Two Fusarium species (F. verticillioides and F. proliferatum) that caused sugarcane pokahh boeng were identified by morphological observation, pathogenicity test, and phylogenetic analysis. Species-specific TaqMan PCR and conventional PCR were designed and optimized to target their rDNA-ITS regions. The sensitivity of the TaqMan PCR was approximately 10 pg of fungal DNA input, which was 1,000-fold over conventional PCR, and successfully detected pokkah boeng in the field-grown sugarcane.
CONCLUSIONS/SIGNIFICANCE: This study was the first to identify two species, F. verticillioides and F. proliferatum, that were causal pathogens of sugarcane pokkah boeng in China. It also described the development of a species-specific PCR assay to detect and confirm these pathogens in sugarcane plants from mainland China. This method will be very useful for a broad range of research endeavors as well as the regulatory response and management of sugarcane pokkah boeng.
由镰刀菌复合体引起的甘蔗梢腐病会导致甘蔗产量大幅损失。因此，迫切需要快速、准确地检测和鉴定该病原体，以管理和预防甘蔗梢腐病的传播。
2012年至2013年期间，从中国五个主要甘蔗产区采集的梢腐病样本中总共分离出101株菌株。通过形态学观察、致病性测试以及基于真菌保守核糖体DNA内转录间隔区（rDNA-ITS）的系统发育分析来鉴定致病病原体。开发了种特异性TaqMan实时荧光定量PCR和常规PCR方法，用于快速、准确地检测甘蔗梢腐病的病原体。还对来自中国的84株镰刀菌分离株以及来自其他病原菌（如黑粉菌和茎点霉）和甘蔗内生真菌（如顶孢霉）的几株分离株评估了PCR检测方法的特异性和灵敏度。
通过形态学观察、致病性测试和系统发育分析，鉴定出两种引起甘蔗梢腐病的镰刀菌（轮枝镰孢菌和层出镰孢菌）。设计并优化了针对其rDNA-ITS区域的种特异性TaqMan PCR和常规PCR。TaqMan PCR的灵敏度约为10 pg的真菌DNA输入量，比常规PCR高1000倍，并成功检测出田间种植甘蔗中的梢腐病。
结论/意义：本研究首次在中国鉴定出两种导致甘蔗梢腐病的病原菌，即轮枝镰孢菌和层出镰孢菌。还描述了一种种特异性PCR检测方法的开发，用于检测和确认中国大陆甘蔗植株中的这些病原体。该方法对于广泛的研究工作以及甘蔗梢腐病的监管应对和管理将非常有用。
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