Development of a Fusarium oxysporum f. sp. melonis functional GFP fluorescence tool to assist melon resistance breeding programmes

Abstract

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (Fom), is one of the most widespread and devastating melon diseases. This vascular disease is caused by the colonization of melon xylem vessels by any of the four Fom races reported (r0, r1, r2 and r1,2, subdivided into r1,2w and r1,2y). The macroscopic evaluation of disease symptoms (disease rating, DR) at several days post‐inoculation (dpi) with Fom spores has been the traditional method to determine the resistance of melon accessions to this fungal pathogen. In this study, one isolate from each Fom race was transformed by Agrobacterium tumefaciens to constitutively express the green fluorescent protein (GFP). Fom‐GFP transformants, as virulent as the corresponding wildtype races, were selected to develop an inoculation assay based on the non‐invasive evaluation of the fluorescence emitted by Fom‐GFP. It was determined that melon root neck was the appropriate area to follow Fom‐GFP and a fluorescence signal rating (FSR) was established in parallel to DR determination. This method allowed the evaluation of GFP signal in the root neck of inoculated melon seedlings at 11–15 dpi. The GFP signal was scored in 62 melon accessions/breeding lines inoculated with different Fom‐GFP, followed by evaluation of the macroscopic DR in the aerial part of melon seedlings at 20–28 dpi. Correlation analysis demonstrated a direct and significant relationship between FSR and DR. This method has shown to be an effective and reliable tool that can assist Fom resistance breeding programmes in melon.

A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea

“…An additional molecular tool that has proven to be useful in other fungi, but is currently unavailable for Armillaria , is the expression of reporter genes. Reporter genes such as luciferase (LUC), (β-glucuronidase (GUS), green fluorescent protein (GFP) and red fluorescent protein (DsRed) and their derivatives have been used to analyse pathogen-host interactions in planta 17 18 19 , facilitate early detection of infection in pathogenicity assays 20 , permit promoter:reporter gene fusions to study gene expression patterns and localisation 21 22 and to assess requirements for heterologous gene expression 23 24 25 26 . Enhanced green fluorescent protein (eGFP), adapted from GFP of the jellyfish Aequorea victoria , is one of the most frequently used reporter genes for expression in Agaricomycetes 21 23 27 28 , owing to its non-destructive visualisation, sensitivity, stability and activity independent of cofactors or additional substrates 29 .…”

Identification of main fungal disease from hydroponic melon in greenhouse

“…melonis is the main pathogenic fungus that attacks melons, especially in greenhouses [20]. Supported by [18], that Fusarium wilt caused by F. oxysporum f. sp. melonis is a disease of melon plants in the greenhouse or in open land which is very detrimental.…”

Fusarium oxysporum f. sp. niveum Molecular Diagnostics Past, Present and Future

“…In the Southeastern United States, Fusarium oxysporum forma specialis niveum (Fon) is widely distributed and causes major yield losses in watermelons, the only host of Fon [10,11]. There are additional FOSC formae speciales that infect watermelon but also infect other crops [12]. Fusarium wilt of watermelon, the disease caused by Fon, results in vascular clogging leading to wilting in one or two infected runners (Figure 1).…”

“…other crops [12]. Fusarium wilt of watermelon, the disease caused by Fon, results cular clogging leading to wilting in one or two infected runners (Figure 1).…”