Mass spore production and inoculation of Calonectria pteridis on Eucalyptus spp. under different environmental conditions

Calonectria pteridis is one of the most important causal agents of Calonectria leaf blight (CLB) of Eucalyptus spp. in Brazil, which is a limiting factor for growth of Eucalyptus plantations. In this study we developed standard procedures for in vitro mass spore production and inoculation of C. pteridis for screening plant species and clones for resistance to CLB. The isolates used in this study were identified using similarity in DNA sequence to reference strains. Among six media tested, glucose asparagine agar induced the highest sporulation production and a significantly enhanced sporulation was found by scraping the aerial mycelium and temporarily submersing the cultures in tap water, followed by drying and additional incubation of the culture. We also demonstrated that the severity of CLB on excised leaves of two Eucalyptus spp. clones increased significantly when the inoculated leaves were incubated in the dark. The optimal temperature for infection was 26ºC when plants were incubated for 48 hours post-inoculation in a mist chamber and then maintained in a greenhouse (25ºC±5ºC) for 50 days. This study identifies environmental conditions to improve spore production and inoculation procedures of C. pteridis for selection of resistant Eucalyptus spp.