Abstract
The filamentous ascomycete Fusarium graminearum (previously also known as Gibberella zeae) is a phytopathogen of grain cereals, reducing crop yield and grain quality. The abilities of sexual reproduction organ-perithecium formation, ascospore formation and discharge are all essential characteristics relevant to F. graminearum disease cycle. Here, we present the details of the protocol to study perithecium formation and ascospore discharge in F. graminearum.
Keywords: Fusarium graminearum, Sexual reproduction, Perithecium formation, Ascospore discharge
Background
The ascomycete fungus Fusarium graminearum is the major causal agent of wheat Fusarium head blight and maize Gibberella stalk rot. This fungus can produce sexual fruiting bodies–perithecia on the surface of colonized host plants, the perithecia overwinters on crop debris and discharge ascospore for next year epidemic (Goswami and Kistler, 2004). Favored by moist and warm conditions, ascospores are forcibly discharged from perithecia and become airborne in air currents as the primary inoculum.This fungus is homothallic; most strains can produce perithecia on carrot agar easily in vitro (Trail and Common, 2000). The microscopic study and a thorough description of perithecia development have been reported with temporal transcriptomic analysis during sexual development of F. graminearum (Trail and Common, 2000; Hallen et al., 2007). After induction of haploid hyphae at 0 h in vitro, dikaryotic cells formed and perithecium initiated at 24 h, young perithecia with central ascogenous cells and developing walls appeared at 48 h. The central ascospore matured at 144 h (Hallen et al., 2007). Studies have been conducted on factors that affect ascospore discharge and have concluded that relative humidity and temperature significantly affect the discharge process, while the light is not essential but it can help (Trail et al., 2002). In this protocol, we outline the method of studying perithecium formation and ascospore discharge in F. graminearum, facilitating the identification of genes that have specific roles in sexual development and disease cycle.
Materials and Reagents
Equipment
Procedure
Note: There is a video presenting the methods of generating perithecia for further studies (https://www.jove.com/video/3895/sexual-development-and-ascospore-discharge-in-fusarium-graminearum). Our protocol was adapted from the video with modifications in fungal culture conditions, aerial mycelium press, perithecia induction conditions and ascospores discharge analysis. The video can be a visual reference to this protocol.
Notes
Data analysis
F. graminearum sexual development capability is often checked with perithecium production assay and ascospores discharge assay. Perithecium density and morphology are parameters for perithecium production assay (Figures 3A and 3B). Ascospores discharge assay includes observations of ascospores discharge or not and the morphology of ascospores (Figures 3C and 3D). Figure 3. The perithecium formation and ascospore discharge analysis of wild-type and mutants with defects in perithecium development or ascospore discharge. A. Perithecium formation in wildtype PH-1. B. The mutant has defects in perithecium development cannot form perithecium on the surface of carrot agar. C. Ascospore discharge was normal in wild-type. D. The mutants had defects in ascospores discharge.
Recipes
Acknowledgments
This protocol was adapted from the previous work (Cavinder et al., 2012). This work was supported by the Natural Science Foundation of China (Grant 31730077) and the Ministry of Agriculture of China (Grant 2016ZX08009-003). The authors declare that there is no conflict of interests.
References
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