Optimization of inoculum dose and humidity conditions to Stemphylium blight for mass scale screening of lentil genotypes

Legumes are a source of nutritional balance in diet providing protein and have importance in the agriculture production system. Nitrogen fixation ability with symbiotic relation of bacteria is an integral role of legumes. They are considered best adopted in prevailing climate change; however, they undergo multiple biotic and abiotic stresses in their agronomic period resulting in heavier yield losses. Stemphylium botryosum is one of the major pathogens of legumes causing blight disease in lentils and other legumes. Different species of legumes have different potential to overcome the devastating pathogenicity of S. botryosum. The study's goal was to optimize the inoculation method to identify effect of inoculum and humidity for disease development in a mass scale screening. Six genotypes of lentils: S6, PARDINA, CDC, ESTON, IG858, and R4 were utilized in the study. Artificial inoculation of S. botryosum was carried out throughout the experiments. Both mycelium and spores were used for the inoculation under different humidity levels (50%, 80%, and 100%). The concentrations of mycelium were adjusted to 1 g/l, 2 g/l, and 5 g/l and spores for inoculum to 50,000 spores/ml, 100,000 spores/ml, and 200,000 spores/ml using sterile water. On a scale of 0 to 10, the disease was rated according to the foliar symptoms and defoliation. To evaluate the severity of the disease, we also computed the relative area under the disease progress curve, or rAUDPC. This work evaluates the infective capacity of S. botryosum mycelium and different inoculum densities to address the problem of restricted generation of S. botryosum conidia for large-scale screening. All genotypes examined showed signs of blight, with the severity increasing with increasing relative humidity (RH). As RH levels rose, disease severity and rAUDPC increased as well, reaching 100% RH. Significantly, S6 and Pardina exhibited consistent resistance, while IG858, R4, and CDC Glamis displayed varying susceptibility. The study highlights the effectiveness of using mycelium grown in liquid Richard's medium as an inoculum for large-scale screenings to assess plant resistance to S. botryosum, proposing a more efficient method over traditional conidia-based approaches. The findings emphasize the necessity of controlled conditions, particularly relative humidity (100% RH) and inoculum density (5 g/l of mycelium), to obtain reliable data for inheritance studies, as unregulated conditions can significantly affect the accuracy of results by masking or amplifying genetic resistance or susceptibility.