Rapid detection and quantification of Fusarium udum in soil and plant samples using real-time PCR

Sukumar Mesapogu,Bandamaravuri Kishore Babu,Achala Bakshi

doi:10.4172/2157-7471.1000107

Abstract

Real-time PCR based detection assay was developed for Fusarium udum, causing vascular wilt of pigeonpea. The Histone-3 gene of F. udum was targeted to design species-specific primers and probe. A comparative study was undertaken to develop a reliable and reproducible procedure for detection and quantification of F. udum from diverse samples. The sensitivity and specificity of oligonucleotides were evaluated through dot blot hybridization, standard and real-time PCR assays. The probe HFUSP showed high degree of sensitivity for DNA obtained from pure cultures of F. udum to that of environmental DNA samples. The qPCR assay specifically differentiated the F. udum from closely related species of Fusarium, other test microbes and environmental samples. The single melting curve at 84.17 and a monomorphic band of 200 bp indicates the specificity and authenticity of the PCR assays. Thus, real-time PCR assay can be used as a rapid and effective procedure that can detect minute amounts of F. udum from complex environments. Therefore, the real-time PCR assay demonstrated in the present study can be successfully used for detection and monitoring of F. udum for early infection and disease epidemiology of vascular wilt of pigeonpea.

Highlights

Fusarium wilt caused by Fusarium udum Butler (1906), alternate teleomorph, (Gibberella indica) [37] is an important biotic constraint in pigeonpea production in Indian subcontinent
All fungal cultures were grown on potato dextrose agar (PDA) plates at 25±2°C while bacterial cultures were maintained on nutrient agar (NA) plates
The primers were specific for F. udum as none of the other tester microbes exhibited any amplification (Figure 4, Table 2)

Summary

Introduction

Fusarium wilt caused by Fusarium udum Butler (1906), alternate teleomorph, (Gibberella indica) [37] is an important biotic constraint in pigeonpea production in Indian subcontinent. Was traditionally based on either symptom on the host or culture dependent isolation of the pathogen from affected host tissue [1]. Identification and detection of pathogenic Fusarium sp. These classical approaches are becoming increasingly problematic because more than one forma specialis may occur on a given host, along with non-pathogenic, common soil and rhizosphere inhabitants [10]. The morphological identification is not feasible to quantify pathogen load in different plant tissues during the growing season or in commodities after harvest. Isolation and culture dependent enumeration may introduce a bias in favor of faster-growing species [38]

Methods

Results

Conclusion