Maleita Carla M, Simões Maria José, Egas Conceição, Curtis Rosane H C, de O Abrantes Isabel M
IMAR-CMA, Department of Life Sciences, University of Coimbra (UC), 3004-517 Coimbra, Portugal.
BIOCANT-Technology Transfer Center, Advanced Sequencing Services, 3060-197 Cantanhede, Portugal.
Plant Dis. 2012 Jun;96(6):865-874. doi: 10.1094/PDIS-09-11-0769-RE.
Meloidogyne hispanica infects many economically important crops worldwide. The accurate identification of this pathogen is essential for the establishment of efficient and sustainable integrated pest management programs. Portuguese M. hispanica isolates were studied by biometrical, biochemical, and molecular characteristics. Biometrical characteristics of M. hispanica females, males, and second-stage juveniles were similar to the original description. Biochemical studies revealed a unique enzyme pattern (Hi4) for M. hispanica esterases that allowed for species differentiation. Molecular analysis of the mtDNA region from COII and 16S rRNA genes resulted in amplification products (1,800 bp) similar to M. hispanica, M. ethiopica, and M. javanica, and the described HinfI was unable to discriminate M. hispanica from the other two species. Analysis of the mtDNA sequences revealed altered nucleotides among the isolates that created new restriction sites for AluI and DraIII. The resulting restriction patterns successfully discriminated between the three species, providing a new tool for Meloidogyne identification. Finally, the phylogenetic relationship between M. hispanica and several Meloidogyne spp. sequences was analyzed using mtDNA, confirming the divergence between meiotic and mitotic species and revealing the proximity of M. hispanica to closely related species. Based on the studies conducted, the application of isozyme or polymerase chain reaction restriction fragment length polymorphism analysis would be a useful and efficient methodology for M. hispanica identification.
西班牙根结线虫感染全球许多具有重要经济价值的作物。准确鉴定这种病原体对于建立高效且可持续的综合虫害管理计划至关重要。通过生物统计学、生物化学和分子特征对葡萄牙的西班牙根结线虫分离株进行了研究。西班牙根结线虫雌虫、雄虫和二龄幼虫的生物统计学特征与最初描述相似。生物化学研究揭示了西班牙根结线虫酯酶独特的酶谱（Hi4），可用于物种区分。对细胞色素氧化酶亚基II（COII）和16S核糖体RNA（rRNA）基因的线粒体DNA（mtDNA）区域进行分子分析，得到了与西班牙根结线虫、埃塞俄比亚根结线虫和爪哇根结线虫相似的扩增产物（1800 bp），并且所描述的HinfI酶无法区分西班牙根结线虫与其他两个物种。mtDNA序列分析揭示了分离株之间的核苷酸变化，这些变化为AluI和DraIII酶创造了新的限制性酶切位点。由此产生的限制性酶切图谱成功地区分了这三个物种，为根结线虫的鉴定提供了一种新工具。最后，利用mtDNA分析了西班牙根结线虫与几种根结线虫属物种序列之间的系统发育关系，证实了减数分裂物种和有丝分裂物种之间的差异，并揭示了西班牙根结线虫与近缘物种的亲缘关系。基于所进行的研究，同工酶或聚合酶链反应-限制性片段长度多态性分析的应用将是一种用于鉴定西班牙根结线虫的有用且高效的方法。
