To determine the pathogenicity of Calonectria species obtained in this study, representative isolates of all Calonectria species isolated from diseased leaves and soils were identified by phylogenetic analyses, and morphological characteristics were selected for inoculation trials. Two Eucalyptus genotypes, E. urophylla × E. tereticornis hybrid genotype CEPT1876 and E. urophylla × E. grandis hybrid genotype CEPT1877 were selected for inoculations. The inoculated Eucalyptus seedlings were three months old and approximately 40 cm tall.

In this study, the inoculations were conducted with both mycelia plug and conidia suspension of selected Calonectria isolates. All the inoculated seedlings were in similar size. In the mycelia plug inoculations, for each Eucalyptus genotype, mycelia plugs of each isolate were inoculated on ten leaves of two to three Eucalyptus seedlings, and ten leaves of other two to three Eucalyptus seedlings treated with sterile MEA plugs were regarded as negative controls. For inoculation, mycelia plugs (5 mm diameter) from 7-day-old MEA cultures were placed upside down on the abaxial surface of the leaflets. In the conidia suspension inoculations, the conidia suspensions for each isolate were prepared using the method described in Graça and co-authors [39] and Wang and Chen [20]. The conidia suspensions prepared for each isolates were measured using a hemocytometer, being the concentration adjusted to 5 × 104 conidia/mL. For each isolate, eight seedlings of each genotype were inoculated by spraying the conidia suspension until the suspension run off the leaves. Sterile water was sprayed onto other eight seedlings as the negative control using the same treatment. To allow sufficient humidity for infection development, the Eucalyptus seedlings inoculated with mycelia plug and conidia suspension, were maintained in plastic chambers (length: 190 cm, width: 90 cm, height: 63 cm) with intermittent water nebulization for 30 s at three-hour intervals and were maintain stable climatic conditions (temperature 24–26 °C; humidity 60–70%) for three days. The experiments using both mycelia plug and conidia suspension were all repeated once using the same methodology.

The plastic chambers were removed three days after inoculation. For mycelia plug inoculations, the length of lesions produced was measured. For conidia suspension inoculations, the disease index (DI) was calculated. Leaf disease severity was assessed by estimating the percentage of lesioned area on each leaf with a scale from 0 to 5, where 0 indicated no lesions, 1 indicated that 1 to 10% area of the leaf was lesioned, 2 indicated that 11 to 25% area of the leaf was lesioned, 3 indicated that 26 to 50% area of the leaf was lesioned, 4 indicated that 51 to 75% area of the leaf was lesioned, and 5 indicated that 76 to 100% area of the leaf was lesioned. The DI was calculated according to Mishra and co-authors [40]. The percentage of lesioned area caused by Calonectria isolate on each leaf of inoculated Eucalyptus seedlings was calculated through the software “Leaf Doctor” [41].

For re-isolations, small pieces of discolored leaf (approximately 0.04 cm2) from the edges of the resultant lesions were cut and placed on 2% MEA at room temperature. Re-isolations were conducted for randomly selected leaves from four randomly selected seedlings of each Eucalyptus genotype for each inoculated isolate, and the randomly selected leaves from all seedlings were inoculated as negative controls. Re-isolations were conducted for both mycelia plug and conidia suspension inoculations. The re-isolated fungi were identified and confirmed by morphological characteristics of culture, macroconidiophore and macroconidia, as well as the disease symptoms produced on the leaves with the original fungi used for inoculations. Statistical analyses were performed using SPSS Statistics 22 software (IBM Corp., Armonk, NY, USA) by one-way analysis of variance (ANOVA) for mycelia plug and conidia inoculation results, respectively. The inoculations were conducted in September, 2020 at the experimental nursery of China Eucalypt Research Centre, GuangDong Province, China.

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