Identification and Regulation of Genes from a Biocontrol Strain of Fusarium oxysporum

Differential display with three time points revealed that thiram altered expression of numerous genes in the biocontrol fungus Fusarium oxysporum CS-20. Of the 101 bands purified from the differential display gel, 86 were successfully cloned, and 64 sequenced. Based on nucleic acid sequences, homology to known products was found using BLASTn for 26 sequences and homology to hypothetical proteins was found for six sequences, also from Gibberella zeae. One band (BM1 24-1) showed homology to an ABC transporter from three different fungi. Because of its association with detoxification functions, the ABC transporter was selected for further study. Mycelia of CS-20 were exposed to 25 lg active ingredient (a.i.) thiram in liquid culture for various times from 0 to 8 h. Quantitative real-time PCR was used to evaluate gene expression. At 30 min after treatment with thiram, the ABC transporter was upregulated 20to 25-fold relative to the control treatment. The ABC transporter was upregulated 15-fold at 1 h after treatment and 10-fold at 2 h. At 8 h after treatment, there was no difference between treated and non-treated for expression of the ABC transporter. Transcription of the gene encoding EST BM1 24-1 is induced in response to thiram treatment and may function in providing resistance in F. oxysporum isolate CS-20 to fungicides and other toxins. Tolerance to toxins may be critical to the successful inclusion of CS-20 in disease control strategies in cropping systems. Introduction Fusarium oxysporum is a soil-inhabiting fungus. Most literature on the identification and function of genes in the genus Fusarium has focused on plant pathogenic strains (e.g., Kistler, 1997; Baayen et al., 1998; Recorbet et al., 2003), including assembling an entire genome [F. graminearum Sequencing Project, Broad Institute of MIT and Harvard (http://www.broad.mit. edu)]. Not all strains of F. oxysporum are pathogenic, and among the saprophytic strains are those which act as biocontrol agents against Fusarium wilt (Fravel et al., 2003a). As biocontrol strains of F. oxysporum and other saprophytic F. oxysporum are morphologically identical to the pathogen, the trait or traits that confer biocontrol ability must be the result of either genetic differences between biocontrol and other F. oxysporum, or differences in expression of genes that may be present in both biocontrol and non-biocontrol strains. Fusarium oxysporum CS-20 is a biocontrol strain that reduces incidence of Fusarium wilt on tomato (Larkin and Fravel, 1999a), muskmelon (Larkin et al., 1999), and basil (Larkin and Fravel, 2002) via a hostmediated response (Larkin and Fravel, 1999b). Preliminary work indicated that one possible difference between two biocontrol strains (including CS-20) and two pathogenic strains of F. oxysporum is in the expression of an ATP-binding cassette (ABC transporter) (Fravel et al., 2003b). In nature, ABC transporters provide protection against toxic compounds such as antibiotics and fungicides (Del Sorbo et al., 2000; Schoonbeek et al., 2002; Stergiopoulos et al., 2002). While strain CS-20 reduced Fusarium wilt under production conditions in several hosts as noted above, failure to achieve biocontrol in the field was possibly associated with the presence of a seed-applied fungicide (Fravel et al., 2005). Hence, genes involved in detoxification of fungicides in CS-20 are of interest from an ecological fitness perspective and for integrating biocontrol with fungicide use in current agricultural practices. This study was undertaken to identify genes expressed in the F. oxysporum biocontrol strain CS-20 and to study the effects of the fungicide thiram on expression of an ABC transporter in CS-20. www.blackwell-synergy.com J. Phytopathology 155, 526–530 (2007) Journal compilation 2007 Blackwell Verlag, Berlin No claim to original US government works doi: 10.1111/j.1439-0434.2007.01271.x