Abstract
Perennial ryegrass is an important temperate grass used for forage and turf worldwide. It forms symbiotic relationships with endophytic fungi (endophytes), conferring pasture persistence and resistance to herbivory. Endophyte performance can be influenced by the host genotype, as well as environmental factors such as seed storage conditions. It is therefore critical to confirm seed quality and purity before a seed is sown. DNA-based methods are often used for quality control purposes. Recently, near-infrared spectroscopy (NIRS) coupled with hyperspectral imaging was used to discriminate perennial ryegrass cultivars and endophyte presence in individual seeds. Here, a NIRS-based analysis of bulk seeds was used to develop models for discriminating perennial ryegrass cultivars (Alto, Maxsyn, Trojan and Bronsyn), each hosting a suite of eight to eleven different endophyte strains. Sub-sampling, six per bag of seed, was employed to minimize misclassification error. Using a nested PLS-DA approach, cultivars were classified with an overall accuracy of 94.1-98.6% of sub-samples, whilst endophyte presence or absence was discriminated with overall accuracies between 77.8% and 96.3% of sub-samples. Hierarchical classification models were developed to discriminate bulked seed samples quickly and easily with minimal misclassifications of cultivars (<8.9% of sub-samples) or endophyte status within each cultivar (<11.3% of sub-samples). In all cases, greater than four of the six sub-samples were correctly classified, indicating that innate variation within a bag of seeds can be overcome using this strategy. These models could benefit turf- and pasture-based industries by providing a tool that is easy, cost effective, and can quickly discriminate seed bulks based on cultivar and endophyte content.