Modified Primers for the Identification of Nonpathogenic Fusarium oxysporum Isolates That Have Biological Control Potential against Fusarium Wilt of Cucumber in Taiwan

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.

此前已有研究证实，对黄瓜无致病力的尖孢镰刀菌（Fusarium oxysporum，Fo）可作为生物防治剂，用于防治台湾地区由黄瓜专化型尖孢镰刀菌（Fusarium oxysporum f. sp. cucumerinum，Foc）引发的黄瓜枯萎病。然而截至目前，学界尚无法通过形态学特征区分具有生防潜力的非致病型尖孢镰刀菌菌株与致病型尖孢镰刀菌种群。尽管可通过生物测定区分这两类种群，但该方法耗力且耗时。本研究利用已发表的通用引物FIGS11/FIGS12，对生防菌株Fo366的核糖体DNA基因间隔区（intergenic spacer，IGS）片段进行聚合酶链式反应（PCR）扩增并测序。基于该IGS序列设计了新引物NPIGS-R，并将其与FIGS11引物配对使用。随后评估了该引物组合对具有生防潜力的非致病型尖孢镰刀菌菌株的扩增特异性。结果显示，优化后的引物组合FIGS11/NPIGS-R可从7株非致病型尖孢镰刀菌菌株中扩增得到500 bp的DNA片段。这5株尖孢镰刀菌菌株在温室生物测定试验中，可延缓黄瓜枯萎病的症状发展。研究人员从土壤与植物组织中分离得到77株尖孢镰刀菌菌株，使用上述优化后的引物组合进行扩增，其中6份样本呈阳性扩增结果。将这6株菌株接种到含菌泥炭藓基质中培养时，未使黄瓜幼苗出现致病症状；后续再向同一植株接种强致病型黄瓜专化型尖孢镰刀菌菌株时，可延缓病害发展。因此，该优化后的引物组合有望用于鉴定对黄瓜无致病力、可作为黄瓜枯萎病生物防治菌剂的尖孢镰刀菌菌株。