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

Microbial Resistance Mechanisms to the Antibiotic and Phytotoxin Fusaric Acid

“…Aspergillus tubingensis was previously sequenced and identified (Crutcher et al 2014). For Fusarium oxysporum, the Fusarium oxysporum specific primer pairs, EF-1A and EF1885R, PHO48F and PHO2034, BT100F and BT1828R, were used to amplify and sequence the elongation factor gene, the phosphate permease gene and beta tubulin (BT) gene, respectively (Ortiz et al 2017). Other primers used include the BT primers BT2a and BT2b (Glass and Donaldson 1995), the internal transcribed spacer region (ITS) primers Fig.…”

Soil-free pathogenicity assay ofFusarium oxysporumf.sp.vasinfectumrace 4 on Pima cotton (Gossypium barbadense) seedlings

“…Since the emergence of Fov4 in the US, a number of studies were performed to improve pathogen detection, population diversity, and distribution surveys (Bell et al 2019;Cianchetta et al 2015;Doan 2014;Ortiz et al 2017). In addition, several disease assay methods were described to test Fov4 virulence against cotton varieties (Cianchetta et al 2015;Ortiz et al 2017;Wang et al 2018).…”

“…Since the emergence of Fov4 in the US, a number of studies were performed to improve pathogen detection, population diversity, and distribution surveys (Bell et al 2019;Cianchetta et al 2015;Doan 2014;Ortiz et al 2017). In addition, several disease assay methods were described to test Fov4 virulence against cotton varieties (Cianchetta et al 2015;Ortiz et al 2017;Wang et al 2018). While there are some modifications, majority of these methods are based on conventional root-cup dip method (Smith et al 1994) or pathogen spore injection into pots where cotton seedling were grown (Ortiz et al 2017).…”

“…In addition, several disease assay methods were described to test Fov4 virulence against cotton varieties (Cianchetta et al 2015;Ortiz et al 2017;Wang et al 2018). While there are some modifications, majority of these methods are based on conventional root-cup dip method (Smith et al 1994) or pathogen spore injection into pots where cotton seedling were grown (Ortiz et al 2017). Disease severity was typically assessed by rating symptoms visually observed on above surface cotton leaves, e.g.…”

“…While we tried to apply these disease assay strategies to our Fov4-cotton interaction research, we faced two major challenges. One was inconsistencies in infection and disease development when inoculating Fov4 spore suspension into cotton seedling pots (Ortiz et al 2017). The other limitation was that we could not investigate infection process occurring at the Fov4-cotton seedling interface to further understand Fov4 virulence at the molecular and cellular level.…”