Specific PCR Detection of <i>Fusarium oxysporum</i> f. sp. <i>vasinfectum</i> California Race 4 Based on a Unique <i>Tfo1</i> Insertion Event in the <i>PHO</i> Gene

Abstract

A highly virulent race 4 (Cal race 4) of Fusarium oxysporum f. sp. vasinfectum was identified in California cotton fields in 2001, and has since been found in increasing numbers of fields. Cal race 4 isolates contain a unique Tfo1 transposon insertion in the PHO gene that was not found in other F. oxysporum f. sp. vasinfectum genotypes. Based on this insertion, a multiplex polymerase chain reaction method was developed to detect the Cal race 4 pathogen. A panel of F. oxysporum f. sp. vasinfectum isolates representing different vegetative compatibility groups (VCG) and DNA sequence types was assembled to test the specificity of the detection method. In all, 16 of 17 Cal race 4 isolates produced a 583-bp amplicon; the other isolate produced a 396-bp amplicon reflecting the absence of the Tfo1 insertion. This isolate was a moderately virulent pathogen among Cal race 4 isolates. In total, 80 other F. oxysporum isolates associated with cotton and 11 other formae speciales of F. oxysporum produced only the 396-bp amplicon. The method also distinguished Cal race 4 isolates from India race 4 isolates and China race 7 isolates, which did not possess the unique Tfo1 insertion but otherwise had identical DNA sequences, and all belong to VCG0114. The method is capable of detecting the pathogen directly from infected stem tissues even before external symptom appears and, thus, provides an effective tool for timely identification of infested fields and seed lots, and should help reduce dissemination of Cal race 4 in the U.S. Cotton Belt.