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Epichloë festucae var. lolii and Epichloë sp. LpTG-3 are filamentous fungal endophytes of perennial ryegrass (Lolium perenne) that have a substantial impact on New Zealand’s agricultural economy by conferring biotic advantages to the host grass. Overall, Epichloë endophytes contribute NZ$200 million to the economy annually, with strain AR37 estimated to contribute NZ$3.6 billion to the New Zealand economy over a 20-year period. This strain produces secondary metabolites, including epoxyjanthitrems, which are a class of indole diterpenes, associated with the observed effects of AR37 on livestock and insect pests. Until very recently, AR37 was intractable to genetic modification but this has changed with the application of CRISPR-Cas9 based gene editing techniques. In this paper, gene inactivation by CRISPR-Cas9 was used to deconvolute the genetic basis for epoxyjanthitrem biosynthesis, including creating an AR37 strain that has been edited to remove the biosynthesis of all indole diterpenes. We show that gene editing of Epichloë can be achieved without off-target events or introduction of foreign DNA (footprint-less) through an AMA1-based plasmid that simultaneously expresses the CRISPR-Cas9 system and selectable marker. Genetic modification events in these transformants were investigated through genome sequencing and in planta chemistry.

The wheat sheath miner, Cerodontha australis Malloch (Diptera: Agromyzidae), is a small leaf-and stem-mining fly that is native to New Zealand and eastern Australia where it feeds on grasses and cereals. Although not considered to be a major insect pest of pastures in New Zealand, it can be abundant during spring and summer in Waikato, New Zealand, and infests tillers of the introduced pasture plant, perennial ryegrass, Lolium perenne L. (Poaceae). Cerodontha australis is not deterred from feeding on L. perenne infected with commercially available strains of the fungal endophyte Epichlo€ e festucae var. lolii, which have been shown to provide protection from a range of other insect pests. In this study, three-pot trials and periodic examination over 18 months of field-collected L. perenne tillers showed that two Epichlo€ e endophyte strains, AR47 and AR48, reduced the survival and feeding damage by C. australis larvae, but there was no evidence of an effect on adult female feeding or oviposition. Compared to other strains of E. festucae var. lolii endophytes tested, plants infected with AR47 and AR48 had fewer and smaller larvae and less larval mining. The number of tillers with mining was reduced in AR47 relative to endophyte-free (Nil) by 57.1% (ranging from 25.0 to 92.5% across trials) and in AR48 by 67.1% (26.3-100%). Larvae in AR47 and AR48 were found further from the base of the tiller where they were less likely to damage the meristem and cause tiller death. The number of larvae and pupae in pseudostems was reduced in AR47 and AR48 compared with Nil by 78.4 and 82.0%, respectively, in the field trial. Very few larvae survived to pupation in AR47 and AR48 plants, with a reduction compared to Nil of over 81.5% in AR47 and over 97% in AR48 in each of the four trials presented here.

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