Modified primers for the identification of nonpathogenic Fusarium oxysporum isolates that have biological control potential against Fusarium wilt of cucumber in Taiwan.

Chaojen Wang,Wenhsin Chung,Yinghong Lin,Yisheng Lin

doi:10.1371/journal.pone.0065093

Chaojen Wang, Wenhsin Chung + Show 2 more

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https://doi.org/10.1371/journal.pone.0065093

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Journal: PloS one	Publication Date: Jun 7, 2013
Citations: 35	License type: CC BY 4.0

Affiliation: National Chung Hsing University, Asian University

Abstract

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.

Highlights

Numerous formae speciales of Fusarium oxysporum (Fo) Schlechtend:Fr. are important pathogens that cause wilt diseases in many different host plants
Among biocontrol agents evaluated for the control of Fusarium wilt caused by Fo formae speciales, the use of nonpathogenic Fo isolates appears to hold much promise
Specificity of the Modified Primer Pair The newly designed primer NPIGS-R combined with FIGS11 was used to assess the amplification of 122 formae speciales isolates, eight different Fusarium spp. isolates and 15 nonpathogenic Fo isolates

Summary

Introduction

Numerous formae speciales of Fusarium oxysporum (Fo) Schlechtend:Fr. are important pathogens that cause wilt diseases in many different host plants. This species is a ubiquitous soil-inhabiting fungus that is a normal constituent of fungal communities in the rhizospheres of plants [1], [2], [3]. Pathogenic Fo isolates showing high host specificity have been classified into more than 150 formae speciales based on plant species and cultivars they infect [4]. Among these formae speciales, F. oxysporum f. Among biocontrol agents evaluated for the control of Fusarium wilt caused by Fo formae speciales, the use of nonpathogenic Fo isolates appears to hold much promise

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