RGB image-based method for phenotyping rust disease progress in pea leaves using R.

BACKGROUND: Rust is a damaging disease affecting vital crops, including pea, and identifying highly resistant genotypes remains a challenge. Accurate measurement of infection levels in large germplasm collections is crucial for finding new resistance sources. Current evaluation methods rely on visual estimation of disease severity and infection type under field or controlled conditions. While they identify some resistance sources, they are error-prone and time-consuming. An image analysis system proves useful, providing an easy-to-use and affordable way to quickly count and measure rust-induced pustules on pea samples. This study aimed to develop an automated image analysis pipeline for accurately calculating rust disease progression parameters under controlled conditions, ensuring reliable data collection. RESULTS: A highly efficient and automatic image-based method for assessing rust disease in pea leaves was developed using R. The method's optimization and validation involved testing different segmentation indices and image resolutions on 600 pea leaflets with rust symptoms. The approach allows automatic estimation of parameters like pustule number, pustule size, leaf area, and percentage of pustule coverage. It reconstructs time series data for each leaf and integrates daily estimates into disease progression parameters, including latency period and area under the disease progression curve. Significant variation in disease responses was observed between genotypes using both visual ratings and image-based analysis. Among assessed segmentation indices, the Normalized Green Red Difference Index (NGRDI) proved fastest, analysing 600 leaflets at 60% resolution in 62 s with parallel processing. Lin's concordance correlation coefficient between image-based and visual pustule counting showed over 0.98 accuracy at full resolution. While lower resolution slightly reduced accuracy, differences were statistically insignificant for most disease progression parameters, significantly reducing processing time and storage space. NGRDI was optimal at all time points, providing highly accurate estimations with minimal accumulated error. CONCLUSIONS: A new image-based method for monitoring pea rust disease in detached leaves, using RGB spectral indices segmentation and pixel value thresholding, improves resolution and precision. It rapidly analyses hundreds of images with accuracy comparable to visual methods and higher than other image-based approaches. This method evaluates rust progression in pea, eliminating rater-induced errors from traditional methods. Implementing this approach to evaluate large germplasm collections will improve our understanding of plant-pathogen interactions and aid future breeding for novel pea cultivars with increased rust resistance.

Genetic Diversity and Population Structure of a Wide Pisum spp. Core Collection.

Peas (Pisum sativum) are the fourth most cultivated pulses worldwide and a critical source of protein in animal feed and human food. Developing pea core collections improves our understanding of pea evolution and may ease the exploitation of their genetic diversity in breeding programs. We carefully selected a highly diverse pea core collection of 325 accessions and established their genetic diversity and population structure. DArTSeq genotyping provided 35,790 polymorphic DArTseq markers, of which 24,279 were SilicoDArT and 11,511 SNP markers. More than 90% of these markers mapped onto the pea reference genome, with an average of 2787 SilicoDArT and 1644 SNP markers per chromosome, and an average LD50 distance of 0.48 and 1.38 Mbp, respectively. The pea core collection clustered in three or six subpopulations depending on the pea subspecies. Many admixed accessions were also detected, confirming the frequent genetic exchange between populations. Our results support the classification of Pisum genus into two species, P. fulvum and P. sativum (including subsp. sativum, arvense, elatius, humile, jomardii and abyssinicum). In addition, the study showed that wild alleles were incorporated into the cultivated pea through the intermediate P. sativum subsp. jomardii and P. sativum subsp. arvense during pea domestication, which have important implications for breeding programs. The high genetic diversity found in the collection and the high marker coverage are also expected to improve trait discovery and the efficient implementation of advanced breeding approaches.

Identification and Characterization of Novel Sources of Resistance to Rust Caused by Uromyces pisi in Pisum spp.

Pea rust is a major disease worldwide caused by Uromyces pisi in temperate climates. Only moderate levels of partial resistance against U. pisi have been identified so far in pea, urging for enlarging the levels of resistance available for breeding. Herein, we describe the responses to U. pisi of 320 Pisum spp. accessions, including cultivated pea and wild relatives, both under field and controlled conditions. Large variations for U. pisi infection response for most traits were observed between pea accessions under both field and controlled conditions, allowing the detection of genotypes with partial resistance. Simultaneous multi-trait indexes were applied to the datasets allowing the identification of partial resistance, particularly in accessions JI224, BGE004710, JI198, JI199, CGN10205, and CGN10206. Macroscopic observations were complemented with histological observations on the nine most resistant accessions and compared with three intermediates and three susceptible ones. This study confirmed that the reduced infection of resistant accessions was associated with smaller rust colonies due to a reduction in the number of haustoria and hyphal tips per colony. Additionally, a late acting hypersensitive response was identified for the first time in a pea accession (PI273209). These findings demonstrate that screening pea collections continues to be a necessary method in the search for complete resistance against U. pisi. In addition, the large phenotypic diversity contained in the studied collection will be useful for further association analysis and breeding perspectives.