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NYMPHSTAR: an accurate high-throughput quantitative method for whitefly (Aleurotrachelus socialis Bondar) resistance phenotyping in cassava


 Background

Whitefly (Aleurotrachelus socialis Bondar) is an important pest causing high economic losses in cassava production systems in the north of South America. It reduces the plant’s photosynthesis by colonizing the cassava leaves and either directly feeding on their phloem sap or excreting substances that allow the growth of sooty mold and the consequent reduction of the photosynthetic area. The deployment of the crop’s natural resistance to this pest is the most effective approach to its management. Phenotypic evaluation to identify germplasm with superior whitefly-resistance (WFR) levels from that showing a whitefly susceptible (WFS) response will benefit from the availability of an accurate high-throughput, quantitative phenotyping method.
Results

We developed an accurate and efficient image-based phenotyping method (Nymphstar) to quantify the total number of third and fourth instar nymphs through red, green, and blue color space (RGB) image analysis as a plugin for ImageJ. Nymphstar estimates both the number of third and fourth instar nymphs and the percentage of leaf area they occupy. We tested 19 cassava genotypes and classified them after data analysis as resistant or susceptible to A. socialis attack. Benchmarking Nymphstar against manual nymph counts performed by a specialist revealed a highly significant correlation between direct nymph counts and those estimated using Nymphstar, which analyzed images and counted nymphs 150 times faster than by manual counts. Full-bench caging for a free-choice assay facilitated our assessment of WFR in the cassava germplasm and early replicated trials in the experimental population and enhanced the efficiency of whitefly (A. socialis) colonization on each cassava genotype to accurately depict the intensity of the resistance/susceptible response under a choice regime, simultaneously reducing human operator bias.
Conclusions

Nymphstar is a fast, accurate image analysis screening tool for the automated counting of nymphs and quantification of leaf area they occupy, allowing for the assessment of cassava resistance to whitefly on a large number of cassava plants in a glasshouse-based assay while avoiding the potential bias normally associated with field assessment and manual counting.