Near-infrared (800–2500 nm; NIR) spectroscopy coupled to hyperspectral imaging (NIR-HSI) has greatly enhanced its capability and thus widened its application and use across various industries. This non-destructive technique that is sensitive to both physical and chemical attributes of virtually any material can be used for both qualitative and quantitative analyses. This review describes the advancement of NIR to NIR-HSI in agricultural applications with a focus on seed quality features for agronomically important seeds. NIR-HSI seed phenotyping, describing sample sizes used for building high-accuracy calibration and prediction models for full or selected wavelengths of the NIR region, is explored. The molecular interpretation of absorbance bands in the NIR region is difficult; hence, this review offers important NIR absorbance band assignments that have been reported in literature. Opportunities for NIR-HSI seed phenotyping in forage grass seed are described and a step-by-step data-acquisition and analysis pipeline for the determination of seed quality in perennial ryegrass seeds is also presented.

Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual Epichloë endophytes have been reported to produce epoxy-janthitrems, LpTG-3 (Lolium perenne Taxonomic Group 3; e.g., NEA12) and LpTG-4 (e.g., E1). Epichloë epoxy-janthitrems are not well understood, the biosynthetic pathway and associated gene complement have not been described and while the literature suggests they are associated with superior protection against pasture insect pests and are tremorgenic in grazing mammals, these properties have not been confirmed using isolated and purified compounds. Whole genome sequence analysis was used to identify candidate genes for epoxy-janthitrem biosynthesis that are unique to epoxy-janthitrem producing strains of Epichloë. A gene, jtmD, was identified with homology to aromatic prenyl transferases involved in synthesis of indole diterpenes. The location of the epoxy-janthitrem biosynthesis gene cluster (JTM locus) was determined in the assembled nuclear genomes of NEA12 and E1. The JTM locus contains cluster 1 and cluster 2 of the lolitrem B biosynthesis gene cluster (LTM locus), as well as four genes jtmD, jtmO, jtm01, and jtm02 that are unique to Epichloë spp. that produce epoxy-janthitrems. Expression of each of the genes identified was confirmed using transcriptome analysis of perennial ryegrass-NEA12 and perennial ryegrass-E1 symbiota. Sequence analysis confirmed the genes are functionally similar to those involved in biosynthesis of related indole diterpene compounds. RNAi silencing of jtmD and in planta assessment in host-endophyte associations confirms the role of jtmD in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I–IV in Epichloë endophytes is proposed.

Methods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as reporter gene products, allowing non-invasive detection in living cells. This study reports the introduction of genes for two fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein, DsRed, into the genomes of two distinct perennial ryegrass (Lolium perenne L.)-associated Epichloë endophyte strains using A. tumefaciens-mediated transformation. Comprehensive characterisation of reporter gene-containing endophyte strains was performed using molecular genetic, phenotypic, and bioinformatic tools. A combination of long read and short read sequencing of a selected transformant identified a single complex T-DNA insert of 35,530 bp containing multiple T-DNAs linked together. This approach allowed for comprehensive characterisation of T-DNA integration to single-base resolution, while revealing the unanticipated nature of T-DNA integration in the transformant analysed. These reporter gene endophyte strains were able to establish and maintain stable symbiotum with the host. In addition, the same endophyte strain labelled with two different fluorescent proteins were able to cohabit the same plant. This knowledge can be used to provide the basis to develop strategies to gain new insights into the host-endophyte interaction through independent and simultaneous monitoring in planta throughout its life cycle in greater detail.

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.