Optimized ascospore ejection method for the evaluation of resistance to Teratosphaeria nubilosa in Eucalyptus

Teratosphaeria nubilosa is the predominant causal agent of Teratosphaeria leaf disease (TLD) in experimental plantations of E. globulus in Brazil. It exhibits slow vegetative growth and lack of sporulation in in vitro culture, making it difficult to obtain enough ascospores for mass inoculation. This study aimed to establish an inoculation method based on ascospore ejection from naturally infected leaves and use it to assess the response to T. nubilosa of eucalypt species and interspecific hybrids. First, the optimal temperature and incubation time for ascospores ejection from naturally infected E. globulus leaves were determined in vitro. Then, these optimal conditions were used to assess the response of eight eucalypt genotypes to T. nubilosa inoculation using ejected ascospores. The infected leaves were placed above (T1), above and under (T2) or under (T3) the plants to be inoculated. Significant differences in disease incidence, disease severity and plant defoliation were observed among genotypes and inoculation treatments. Plants exhibited higher incidence, more severe symptoms and more defoliation when exposed to T2. E. globulus and E. globulus × E. nitens clones were the most susceptible whereas an E. benthamii clone was immune. The sources of resistance identified in this work can be used in hybridization programmes with E. globulus to obtain interspecific hybrids expressing resistance to T. nubilosa and retaining the desirable traits for planting in temperate climates. The inoculation method optimized in this work could be used for small and medium-scale screening of Eucalyptus spp. resistant to TLD. However, periodic validation of the species and genotype of the fungus associated with the naturally infected leaves used for inoculation and confirmation of the resistant phenotype of the selected eucalypt clones or progenies through experimental replications over time must be conducted.