Providing inoculum for Didymella bryoniae studies: the effect of light spectrum and storing at low temperature.

M C S Virtuoso,L T Braz,P F Vargas,E M Silva,T S Valente,R C Panizzi,E H C Silva

doi:10.1590/1519-6984.253436

Abstract

The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media (PDA, V8, ML, and PDAB) and, to evaluate fungus' survivability stored at -20°C over time. The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media. However, there was considerable variation in the amount of each structure between the different culture media. The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover, the culture medium composition influences the type of fungal structure produced, as well as spores' size and quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months without loss of viability.

Highlights

The Didymella bryoniae (Auersw.) Rehm, synonym Stagonosporopsis cucurbitacearum (Fr.) [anamorph Phoma cucurbitacearum (Fr.) Sacc (=Ascochyta cucumis Fautrey & Roum)] (Mao et al, 2020), is an ascomycete that causes the gummy stem blight (GSB) in several cucurbits
There was no significant interaction between the culture medium and the type of light used to promote in vitro sporulation of D. bryoniae
The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium

Summary

Introduction

The Didymella bryoniae (Auersw.) Rehm, synonym Stagonosporopsis cucurbitacearum (Fr.) [anamorph Phoma cucurbitacearum (Fr.) Sacc (=Ascochyta cucumis Fautrey & Roum)] (Mao et al, 2020), is an ascomycete that causes the gummy stem blight (GSB) in several cucurbits. GSB-related studies often require the combination of inoculations in a greenhouse and subsequent field tests (Gusmini et al, 2003). Several studies evaluating the best condition for D. bryoniae sporulation in vitro have been reported, testing different culture medium (CM) combinations (Gusmini et al, 2003; Hassan et al, 2018; Leão et al, 2012; Nga et al, 2010; Skarshaug, 1981; Somai et al, 2002), ultraviolet (UV) light (Kwon et al, 1997; Lee, 1982; Tsay et al, 1990; Tsutsumi and Silva, 2004; Wolukau et al, 2007; Zhang et al, 2017), and temperature and pH (Bhat et al, 2009). Despite the vast literature on the subject, there is still no consensus among researchers on the main factors influencing in vitro D. bryoniae sporulation, with frequent disagreement between studies

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Conclusion