Journal of Applied Microbiology (2014) 116, 942-954

A. Moradi, M.A. Almasi, H. Jafary and J. Mercado-Blanco (2014)
A novel and rapid loop-mediated isothermal amplification assay for the specific detection of Verticillium dahliae
Journal of Applied Microbiology 116 (4), 942-954
Abstract:
Aims
In this study, a loop-mediated isothermal amplification (LAMP) assay has been developed and evaluated for the rapid and sensitive detection of Verticillium dahliae Kleb., the causal agent of vascular wilts in many economically important crops.
Methods and Results
LAMP primers were designed based on a previously described RAPD marker, and the LAMP assay was applied for direct detection of V. dahliae grown on medium and from soil samples without DNA purification steps (direct-LAMP). Thirty-two agricultural soil samples from various olive orchards were collected, and the presence of pathogen was detected by LAMP, direct-LAMP and nested-PCR methods. The LAMP methodology could successfully detect V. dahliae with high specificity, and cross-reaction was not observed with different pathogenic and nonpathogenic fungi and bacteria. The LAMP assay was capable of detecting a minimum of 500 and 50 fg of purified target DNA per reaction of V. dahliae ND and D pathotypes, respectively. In contrast, nested-PCR could only detect 5 pg reaction⁻¹ for both pathotypes. In artificially infested soil samples, the LAMP method detected 5 microsclerotia per gram of soil. Conversely, nested-PCR assay detected 50 microsclerotia g⁻¹ soil. The detection ratios of LAMP and direct-LAMP protocols were better (26 and 24 positive samples out of 32 agricultural soils analysed, respectively) than that obtained for nested-PCR method (22 positive results). Moreover, direct-LAMP yielded positive detection of V. dahliae in agricultural soil samples within 60-80 min.
Conclusions
The newly developed LAMP method was proved to be an effective, simple and rapid method to detect V. dahliae without the need for either expensive equipment or DNA purification.
Significance and Impact of Study
This technique can be considered as an excellent standard alternative to plating and nested-PCR assays for the early, sensitive and low-cost detection of V. dahliae as well as other soilborne pathogens in the field.
(The abstract is excluded from the Creative Commons licence and has been copied with permission by the publisher.)
Link to article at publishers website
Database assignments for author(s): Jesús Mercado-Blanco

Research topic(s) for pests/diseases/weeds:
molecular biology - genes

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Pest and/or beneficial records:

Beneficial	Pest/Disease/Weed	Crop/Product	Country	Quarant.
	Verticillium dahliae