Identification of binucleate Rhizoctonia as a contaminant in pot cultures of arbuscular mycorrhizal fungi and development of a PCR-based method of detection

Abstract

Binucleate Rhizoctonia (BNR) was isolated (isolate CFM1) from cabbage grown in soil from pot cultures of the arbuscular mycorrhizal (AM) fungus, Glomus mosseae, grown on subterranean clover, and was identified as AG-Bo by means of morphological and molecular characteristics. A PCR-based assay was developed to detect BNR in roots and soils of pot cultures of AM fungi. Two oligonucleotide primers (CF1f and CF2r) were designed from the ITS region of the rDNA sequence of BNR AG-Bo isolate CFM1. These allowed amplification of a specific fragment (438 bp). Specificity of the primers was tested against several isolates of various anastomosis groups (AGs) of BNR, other related and unrelated fungal species, and roots not infected or infected with fungi. The primers amplified DNA of isolates of both BNR AG-Bo and AG-A, and were used to detect them in roots in the presence or absence of AM fungi. Nested PCR amplification using CF1f and CF2r primers has advantages over microscopic methods for detection of BNR AG-Bo or AG-A on infected roots and soils of AM pot cultures and provides a powerful tool for detection of contamination of these pot cultures.