Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan.
PLoS One. 2013 Jun 7;8(6):e65093. doi: 10.1371/journal.pone.0065093. Print 2013.
Previous investigations demonstrated that Fusarium oxysporum (Fo), which is not pathogenic to cucumbers, could serve as a biological control agent for managing Fusarium wilt of cucumber caused by Fo f. sp. cucumerinum (Foc) in Taiwan. However, thus far it has not been possible to separate the populations of pathogenic Fo from the nonpathogenic isolates that have biological control potential through their morphological characteristics. Although these two populations can be distinguished from one another using a bioassay, the work is laborious and time-consuming. In this study, a fragment of the intergenic spacer (IGS) region of ribosomal DNA from an Fo biological control agent, Fo366, was PCR-amplified with published general primers, FIGS11/FIGS12 and sequenced. A new primer, NPIGS-R, which was designed based on the IGS sequence, was paired with the FIGS11 primer. These primers were then evaluated for their specificity to amplify DNA from nonpathogenic Fo isolates that have biological control potential. The results showed that the modified primer pair, FIGS11/NPIGS-R, amplified a 500-bp DNA fragment from five of seven nonpathogenic Fo isolates. These five Fo isolates delayed symptom development of cucumber Fusarium wilt in greenhouse bioassay tests. Seventy-seven Fo isolates were obtained from the soil and plant tissues and then subjected to amplification using the modified primer pair; six samples showed positive amplification. These six isolates did not cause symptoms on cucumber seedlings when grown in peat moss infested with the isolates and delayed disease development when the same plants were subsequently inoculated with a virulent isolate of Foc. Therefore, the modified primer pair may prove useful for the identification of Fo isolates that are nonpathogenic to cucumber which can potentially act as biocontrol agents for Fusarium wilt of cucumber.
先前的研究表明，对黄瓜不致病的尖孢镰刀菌（Fo）可以作为生物防治剂，用于防治台湾黄瓜枯萎病（由 Fo f. sp. cucumerinum（Foc）引起）。然而，到目前为止，还无法通过形态特征将具有生物防治潜力的致病性 Fo 种群与非致病性分离株分开。尽管可以使用生物测定法将这两个种群区分开来，但这项工作既费力又耗时。在这项研究中，使用已发表的通用引物 FIGS11/FIGS12 对 Fo 生物防治剂 Fo366 的核糖体 DNA 基因间隔区（IGS）区域的一个片段进行了 PCR 扩增并进行了测序。根据 IGS 序列设计了一个新的引物 NPIGS-R，并与 FIGS11 引物配对。然后评估了这些引物对具有生物防治潜力的非致病性 Fo 分离株 DNA 的特异性。结果表明，改良的引物对 FIGS11/NPIGS-R 从七个非致病性 Fo 分离株中的五个扩增出 500bp 的 DNA 片段。这五个 Fo 分离株在温室生物测定试验中延迟了黄瓜枯萎病的症状发展。从土壤和植物组织中获得了 77 个 Fo 分离株，然后使用改良的引物对进行扩增；六个样品显示出阳性扩增。当将这些分离株种植在受污染的泥炭藓中时，它们不会引起黄瓜幼苗出现症状，并且当随后用 Foc 的强毒分离株接种同一植物时，它们会延迟疾病的发展。因此，改良的引物对可能有助于鉴定对黄瓜不致病且可能作为黄瓜枯萎病生物防治剂的 Fo 分离株。
