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Intergenic Spacer--RFLP Analysis and Direct Quantification of Australian Fusarium oxysporum f. sp. vasinfectum Isolates from Soil and Infected Cotton Tissues

Fusarium wilt of cotton, caused by Fusarium oxysporum f. sp. vasinfectum, can have devastating effects on the vascular system of cotton plants and is a major threat to cotton production throughout the world. Accurate characterization and improved detection of these pathogenic isolates is needed for the implementation of a disease prevention program and the development of disease management strategies. Polymerase chain reaction (PCR) amplification of the ribosomal intergenic spacer (IGS) regions combined with digestion with three restriction enzymes (AluI, HaeIII, RsaI) resulted in three unique restriction profiles (IGS-restriction fragment length polymorphism [RFLP] haplotypes) for Australian F. oxysporum f. sp. vasinfectum isolates, which were capable of distinguishing them from other formae speciales of F. oxysporum. Furthermore, a portion of the IGS region flanking the 5′ end was sequenced and single nucleotide polymorphisms (SNPs) were revealed. Using these sequence data, two specific real-time PCR-based assays were developed for the absolute quantification of genomic DNA from isolates obtained from soil substrates and infected cotton tissues. Standard curves of real-time PCR-based assays showed a linear relation (R² = 0.993 to 0.994) between log values of fungal genomic DNA and real-time PCR cycle thresholds. Using these assays, it was possible to detect fungal DNA as low as 5 pg/μl. The detection sensitivity for inoculum added to sterile soils was lower than 10⁴ conidia/g soil. In plant samples, the quantified fungal DNA varied from 30 pg to 1 ng/100 ng of total plant genomic DNA.