Insect bioactive capabilities of Epichloë festucae var lolii AR48 infected Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand

Figures printed with permission from respective publishers.As the modern world expands and develops, new innovative methodologies for more efficient and environmentally friendly agricultural practices are required. Loss of crops through abiotic (e.g. drought) and biotic (e.g. herbivory) stresses has a major effect on the success of an agricultural industry. For animal production pasture crops are a key aspect of animal husbandry and directly affects yield and health. Symbiotic fungi belonging to the genus Epichloë form associations with cool season forage grasses and have been exploited as a new innovative method for insect pest management. Ryegrass infected with the asexual E. festucae var lolii strain AR48 has insect bioactivity against both the stem boring fly (SBF-Ceradontha australis) and cutworm moth caterpillar (CC -Agrotis ipsilion). The bioactive/s targeting both insects is currently unknown. The aim of this thesis was to identify the gene/s and/or bioactive/s present in AR48 infected ryegrass that have bioactivity against the SBF and/or CC. Two approaches were taken; the known insect bioactive secondary metabolite pathways in Epichloë were investigated in AR48 through bioinformatics and mass spectrometry, and the gene ‘makes caterpillars floppy’ (mcf), encoding an insect toxin like protein, was investigated through reverse genetics and insect bioactivity trials. A new indole diterpene compound (IDT) was identified in AR48 infected plant material and this compound was absent in other Epichloë strains that do not have SBF and CC bioactivity. The same mcf gene allele as that present in the E. typhina mcf model, previously identified as having CC bioactivity, is present and predicted to be functional in AR48. The other Epichloë strains also have mcf genes predicted to be functional, however the mcf allele is different to the bioactive E. typhina mcf model. Overall, this project was able to identify a new IDT compound with potential insect bioactivity as well as identify two Epichloë mcf gene alleles that potentially have differing insect bioactivities

How does Epichloë festucae avoid the host defence response? : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand

Epichloë festucae is a filamentous fungus, which forms symbiotic associations with aerial tissues of Lolium and Festuca grass species. Chitin, a polymer of N-acetyl-Dglucosamine, is an important component of the fungal cell wall and a well-known pathogen associated molecular pattern (PAMP). Chitin promotes pathogen-triggered immunity (PTI) upon hydrolysis with plant chitinases and release of chitin oligomers. Therefore, to establish a stable and successful symbiosis, the endophyte needs to remain ‘hidden’ from the host immune system or actively suppress it. Confocal laser scanning microscopy (CLSM)-based analysis of leaf tissue infected with the E. festucae wild type strain and infiltrated with the chitin-specific molecular probe, WGA-Alexa Fluor-488, showed that only the septa of endophytic hyphae bound this probe while the entire cell wall was labelled in epiphyllous hyphae confirming previous observations that hyphal cell wall chitin is either masked or remodelled in endophytic hyphae. The aims of this project were (i) to test whether E. festucae LysM-containing proteins have a role in binding to or sequestering cell wall chitin oligomers and thereby preventing PAMPtriggered immunity and (ii) to analyse the composition of the cell wall of endophytic and epiphytic hyphae. An analysis of the E. festucae genome identified seven genes encoding proteins with LysM domains. Expression of two of these genes, lymA and lymB, increased in planta compared to in culture. Interestingly, both are divergently transcribed from chitinase encoding genes (chiA and chiB respectively), which also have increased expression in planta. Single gene deletion mutants of lymA, lymB, chiA and chiB as well as a double gene deletion ΔlymA/B were generated, and their plant interaction phenotype analysed. Plants infected with DlymA, DlymB or DchiA had the same plant-interaction phenotype as wild type whereas ΔchiB and ΔlymA/B mutants had defects in hyphal growth within the leaves. Analysis of hyphal cell wall structure using Chitin Binding Protein (CBP) and chitosan (CAP (Chitosan Affinity Protein) and OGA-488)-specific eGFP-based biosensors suggest that cell wall chitin is converted to chitosan in endophytic hyphae. This structural change is consistent with a lack of a defence response when E. festucae forms a mutualistic symbiotic association with L. perenne. Three E. festucae chitin deacetylase genes were identified (cdaA, cdaB and cdaC), and gene expression analysis showed cdaA expression is significantly increased in planta compare to in culture. Functional analysis of cdaA revealed that although plants infected with the ΔcdaA mutant had a similar whole plant interaction phenotype as wild type, they had an abnormal cellular phenotype. Patches of chitin were exposed along the endophytic hyphae confirming this mutant was unable to convert chitin to chitosan. However, hyphae in these plants still labelled with the chitosan biosensor OGA-488 demonstrating that despite the deletion of the cdaA, the hyphal cell wall of endophytic hyphae still contain chitosan suggesting that another chitin deacetylase, possibly CdaB has a redundant function in E. festucae. Collectively these results show that lymA, lymB and chiB are required for establishment of the symbiosis between E. festucae and L. perenne. In addition, this study shows that chitin is converted to chitosan in the hyphal cell wall of endophytic hyphae during the infection and colonisation of the host. The E. festucae chitin deacetylase gene cdaA is also essential for proper hyphal growth in planta and the symbiotic interaction

Drought tolerance of perennial ryegrass (Lolium perenne L.) and the role of Epichloë endophyte : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand

Perennial ryegrass is the most important grass species in New Zealand. Due to climate change, drought will become more severe and frequent in New Zealand, which makes it increasingly important to improve drought tolerance of perennial ryegrass. There are many ryegrass cultivars in the seed market; however, very limited information is available about drought tolerance of these cultivars. Therefore, the first aim of this thesis was to compare drought tolerance of several market-leading perennial or long-rotation ryegrass cultivars in order to provide cultivar information for pastoral industry. Epichloë festucae var. lolii fungal endophyte naturally colonises perennial ryegrass. Reported effects of endophyte on drought tolerance of the host perennial ryegrass are multifarious. Therefore, the second aim of this thesis was to investigate effects of endophyte on drought tolerance of perennial ryegrass comprehensively. Two main experiments were conducted in this PhD project. In the first experiment, endophyte-free (E–) and endophyte-infected (E+) cloned plants of seven perennial or long-rotation ryegrass cultivars (Grasslands Commando, Ceres One50, Banquet II, Alto, Bealey, Trojan and Avalon), an un-released elite perennial ryegrass line (URL) and one Mediterranean tall fescue cultivar (Grasslands Flecha) were subjected to a cycle of drought and rehydration from December 2012 to May 2013 while other clones of the same plants were irrigated. In the second experiment, two perennial ryegrass cultivars One50 and Commando infected with and without the AR37 endophyte were subjected to a glasshouse experiment. Eight genotypes of each cultivar with and without endophyte infection were either under irrigation or withheld irrigation for two weeks and then rehydrated for one month. A series of plant morphological and physiological responses were measured in each experiment. In the rainout shelter experiment, it was found that Flecha tall fescue was more tolerant to drought than ryegrass cultivars, but this was attributed to its small plant size induced by the partial summer dormancy. Introducing germplasm from Mediterranean areas would be an option to improve drought tolerance of perennial ryegrass in New Zealand. Among evaluated ryegrass cultivars, Banquet II was relatively more drought tolerant than other cultivars, which was also mainly due to its small plant size. In the glasshouse experiment, it was found that Spanish germplasm based One50 was more drought tolerant than „Mangere? ecotype based Commando, suggesting that Spanish germplasm has conferred enhanced drought tolerance to perennial ryegrass in New Zealand. Under both irrigated and non-irrigated conditions, endophyte infection reduced the herbage yield, decreased the relative water content, osmotic potential and stomatal conductance (as indicated by carbon isotope discrimination) and increased the proline concentration of the host compared to E– plants. Also, a majority of these effects were more pronounced in the URL (infected with AR37) and One50 (infected with AR1). It was concluded that E+ plants are at a disadvantage compared to E- plants when insect pressure is artificially controlled, no matter whether the water availability is high or low

Viability of endophytic fungus in different perennial ryegrass (Lolium perenne) varieties kept in different storage conditions : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University, Manawatū, New Zealand

Epichloë endophytes form symbiotic relationships with cool-season grasses of the Pooideae family and are known to synthesise a range of bio-protective alkaloids. These alkaloids can provide the grass host with benefits for greater survival including; deterrence of herbivorous pests, increased persistence, better livestock health and protection from abiotic stressors. The commercialisation of novel endophytes is on the increase, and it is important to ensure the survival of the endophyte is maintained so their benefits can be realised. This study examined the effects of different storage conditions on the viability of three commercial novel endophytes (AR1, AR37 and NEA2/6) and one pre-commercial novel endophyte (815). The different storage conditions were the top of a warehouse, the bottom of a warehouse and a temperature and humidity controlled cool store to simulate current commercial seed storage environments. The viability of different endophytes decreases independently of grass seed germination (p = NS) however there are many factors influencing the endophyte survival. Over the one year storage period there were significant interactions between endophyte x ploidy (host), endophyte x location and endophyte x ploidy (host) x location. The pre-commercial endophyte, 815, had the largest reduction in viable endophyte when stored outside of the controlled cool store dropping 70 percentage points at the top of the warehouse, compared with AR37 (12 percentage points), AR1 (16 percentage points), and NEA2/6 (46 percentage points) (p<.001, LSD = 15.9). In the cool store there was no significant decrease in any of the treatments. As more novel endophyte/grass combinations are released for commercial sale it is important to test each for compatibility and performance post-storage. The results of this study recommend controlled low-temperature, low-humidity storage to maintain endophyte viability. Keywords: Endophyte, Epichloë, perennial ryegrass, Lolium perenne, storag

A conserved signalling network regulates Epichloë festucae cell-cell fusion and the mutualistic symbiotic interaction between E. festucae and Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand

Content redacted from thesis due to copyright reasons: Figure 1.1 Page 4 Schardl, C. L. (2001) Epichloë festucae and related mutualistic symbionts of grasses. Fungal Genet. Biol., 33, 69-82. http://www.sciencedirect.com/science/article/pii/S1087184501912757 Schardl, C. L., Young, C. A., Hesse, U., Amyotte, S. G., Andreeva, K., Calie, P. J., et al . (2013) Plant-symbiotic fungi as chemical engineers: Multigenome analysis of the Clavicipitaceae reveals dynamics of alkaloid loci. Plos Genetics, 9(2), e1003323. http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1003323 Christensen, M. J., Bennett, R. J., Ansari, H. A., Koga, H., Johnson, R. D., Bryan, G. T., et al. (2008) Epichloë endophytes grow by intercalary hyphal extension in elongating grass leaves. Fungal Genet. Biol., 45, 84-93. http://www.sciencedirect.com/science/article/pii/S1087184507001259 Becker, M., Becker, Y., Green, K. & Scott, B. (2016) The endophytic symbiont Epichloë festucae establishes an epiphyllous net on the surface of Lolium perenne leaves by development of an expressorium, an appressorium-like leaf exit structure. New Phytol., 211, 240-254. http://onlinelibrary.wiley.com/doi/10.1111/nph.13931/abstract Figure 1.2 Page 6 Scott, B. (2015) Conservation of fungal and animal nicotinamide adenine dinucleotide phosphate oxidase complexes. Mol Microbiol. 95(6):910-3. doi: 10.1111/mmi.12946. http://onlinelibrary.wiley.com/doi/10.1111/mmi.12946/abstract;jsessionid=087348D78C98893FF6F685B4A3D31179.f01 t03. Figure 1.3 Page 9 Haruo Saito & Kazuo Tatebayashi (2004) Regulation of the Osmoregulatory HOG MAPK Cascade in Yeast. The Journal of Biochemistry. 136 (3): 267-272. http://jb.oxfordjournals.org/content/136/3/267.abstract Figure 1.4 Page 10 Eaton, C., Mitic, M., Scott., B (2012) Signalling in the Epichloë festucae: Perennial Ryegrass Mutualistic Symbiotic Interaction. Signaling and Communication in Plant Symbiosis, pp.143-181. DOI: 10.1007/978-3-642-20966-6_7. http://link.springer.com/chapter/10.1007%2F978-3-642-20966-6_7 Figure 1.5 Page 14 Kück, U., Pöggeler, S., Nowrousian, M., Nolting, N., Engh, I., (2009) Sordaria macrospora, a model system for fungal development. In: Anke, T., Weber, D. (Eds.), The Mycota XV, Physiology and Genetics. Springer, Berlin/Heidelberg, pp. 17–39. http://homepage.rub.de/minou.nowrousian/Reprints/2009Mycota.pdf Figure 1.6 Page 16 Coppin, E., Debuchy, R., S. Arnaise, & Picard, M. (1997) Mating types and sexual development in filamentous ascomycetes. Microbiology and Molecular Biology Reviews, 61, 411-428. http://mmbr.asm.org/content/61/4/411.abstract Aldabbous, M. S., Roca, M. G., Stout, A., Huang, I. C., Read, N. D. & Free, S. J. (2010) The ham-5, rcm-1 and rco-1 genes regulate hyphal fusion in Neurospora crassa. Microbiology, 156, 2621- 2629. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068686/pdf/2621.pdf Silar, P., Lalucque, H., & Vierny, C. (2001) Cell degeneration in the model system Podospora anserina. Biogerontology, 2, 1–17. https://www.ncbi.nlm.nih.gov/pubmed/11708613 Jamet-Vierny, C., Debuchy, R., Prigent, M. & Silar, P. (2007) IDC1, a pezizomycotina-specific gene that belongs to the PaMpk1 MAP kinase transduction cascade of the filamentous fungus Podospora anserina. Fungal Genet. Biol., 44, 1219-1230. http://fulltext.study/preview/pdf/2181591.pdf Figure 1.7 page 20 Fleißner, A., Leeder, A. C., Roca, M. G., Read, N. D. & Glass, N. L. (2009) Oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion. Proc. Natl. Acad. Sci. USA, 106, 19387-19392. http://www.pnas.org/content/106/46/19387.long Figure 1.8 page 22 Kück, U., Beier, A. M. & Teichert, I. (2016) The composition and function of the striatin-interacting phosphatases and kinases (STRIPAK) complex in fungi. Fungal Genet. Biol., 90, 31-38. http://www.sciencedirect.com/science/article/pii/S1087184515300347 Figure 4.8 page 143 Frey, S., Lahmann, Y., Hartmann, T., Seiler, S. & Pöggeler, S. (2015) Deletion of Smgpi1 encoding a GPI-anchored protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora. Mol. Microbiol., 97, 676-697. http://onlinelibrary.wiley.com/doi/10.1111/mmi.13054/suppinfoEpichloё festucae is a filamentous fungus that forms a mutually beneficial symbiotic association with Lolium perenne. The NADPH oxidase complex components noxA, noxR and racA, the transcription factor proA, and the cell wall integrity (CWI) MAP kinases, mkkA and mpkA, are required for mutualistic E. festucae-L. perenne associations and cell-cell fusion. Homologues of these genes in Neurospora crassa, Sordaria macrospora and Podospora anserina are required for cell-cell fusion and sexual fruiting body maturation, thereby establishing a link between self signalling and hyphal network formation in the E. festucae-L. perenne symbiosis. In Podospora anserina, IDC2 and IDC3 are required for cell-cell fusion, crippled growth and fruiting body formation. In S. macrospora and N. crassa, components of the STRIPAK complex regulate cell-cell fusion and fruiting body formation. The aim of this project was to test if E. festucae homologues of IDC2 and IDC3, and the STRIPAK complex protein MOB3, named SymB, SymC and MobC, respectively, are also required for cell-cell fusion and plant symbiosis. Gel shift assays showed the promoters of symB and symC are targets for the transcription factor ProA. In culture, the frequency of cell-cell fusion of ΔmobC was reduced, but in ΔsymB and ΔsymC mutants, totally abolished. All three mutants hyperconidiated and formed intra-hyphal hyphae. Plants infected with these mutants were severely stunted and hyphae exhibited proliferative growth and increased colonisation of the intercellular spaces and vascular bundles. Expressoria formation, structures allowing colonisation of the leaf surface, was reduced in ΔmobC, and abolished in ΔsymB and ΔsymC mutants. Microscopy analyses showed SymB-GFP and SymC-mRFP1 co-localise to the plasma membrane and septa. SymC also localised to highly dynamic punctate structures. Although ΔsymB and ΔsymC phenotypes are identical to ΔmpkA, and the E. festucae pheromone response pathway scaffold ΔidcA mutants, MpkA and MpkB phosphorylation and cellular localisation was unchanged compared to wild-type. Using yeast-two-hybrid assays, an interaction between SymC and the STRIPAK complex associated protein GPI1 was demonstrated. Collectively these results show that MobC, SymB and SymC are required for E. festucae cell-cell fusion and host symbiosis. It is proposed that SymB and SymC interact to form a sensor complex at the cell wall which regulates cell-cell fusion in culture and hyphal network development in planta

Fungal alkaloid occurrence during seedling establishment and early growth in Lolium perenne seedlings infected with Epichloë festucae var. lolii and the influence of adult Argentine stem weevil (Listronotus bonariensis) feeding on alkaloid concentrations : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Agriculture Science at Massey University, Palmerston North, New Zealand

Fungal endophytes of the genus Epichloë often form stable, symbiotic, and mutualistic relationships with grasses of the Pooideae, including perennial ryegrass (Lolium perenne L.). The endophyte provides various benefits to its grass host, through the production of secondary metabolites, which are exploited in New Zealands pastoral agriculture systems. The endophyte can give its host grass an ecological advantage in certain challenging environments, such as during seedling establishment, where young plants are especially vulnerable to insect predation, such as feeding by adult Argentine stem weevil (ASW, Listronotus bonariensis). This thesis focuses on understanding the alkaloid concentrations that occur in endophyte-infected perennial ryegrass seedlings during the early establishment phase. A glasshouse experiment was conducted in which fungal alkaloid concentrations (peramine, lolitrem B, ergovaline, and epoxy-janthitrems) were measured in perennial ryegrass seedlings infected with Epichloë festucae var. lolii strains AR1, AR37, NEA2, and NZCT for 69 days after sowing. From the data it is inferred that an initial translocation of alkaloids stored in seed during maturation into the developing shoot of the germinating seedling occurs, followed by a period of alkaloid dilution due to seedling expansion, and finally production of newly metabolised alkaloids in the plant. Alkaloid concentration were found to peak in 8–10 day old seedlings, giving the seedling a “kick start” in protection of the emerging seedling from adult ASW feeding during the first 11 days after sowing. The influence of adult ASW feeding on alkaloid concentrations in endophyte-infected perennial ryegrass seedlings was also tested. The study demonstrated that adult ASW feeding can influence alkaloid production, although peramine, the main alkaloid responsible for ASW deterrence was not significantly affected. Findings from this thesis improve understanding of the role of fungal alkaloids in endophyte-infected perennial ryegrass seedlings during establishment, and help explain results from earlier studies describing seedling susceptibility to adult ASW

Epigenetic regulation of Epichloë festucae secondary metabolite biosynthesis and symbiotic interaction with Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand

Histone methylation is one of several epigenetic layers for transcriptional regulation. Most studies on the importance of this histone modification in regulating fungal secondary metabolite gene expression and pathogenicity have focussed on the role of histone methyltransferases, while few studies have focussed on the role of histone demethylases that catalyse the reversal of the modification. Epichloë festucae (Ascomycota) is an endophyte that forms a mutualistic interaction with perennial ryegrass. The fungus contributes to the symbiosis by the production of several classes of secondary metabolites, these have anti-insect and/or anti-mammalian activity. The EAS and LTM clusters in E. festucae are located subtelomerically and contain the biosynthetic genes for two of these important metabolites which are only synthesised in planta. Thus, in the host plant these genes are highly expressed, but they are tightly silenced in culture conditions. Previous study has shown that histone H3K9 and H3K27 methylation and their corresponding histone methyltransferases are important for this process. In this study, the role of histone lysine demethylases (KDMs) in regulating these genes and the symbiotic interaction is described. Eight candidate histone demethylases (Jmj1-Jmj8) were identified in E. festucae, among these proteins are homologues of mammalian KDM4, KDM5, KDM8, JMDJ7, and N. crassa Dmm-1. The genes for the proteins were overexpressed in E. festucae and histone methylation levels were determined in the strains. Overexpression of the genes was not observed to cause any change to the culture and symbiotic phenotypes of the fungus. Western blot analysis subsequently identified one of the proteins, KdmB, as the histone H3K4me3 demethylase. Further analysis by ChIP- and RT-qPCR showed that demethylation of H3K4me3 by KdmB at the eas/ltm genes is crucial for the activation of these genes in planta. The full expression of several other telomeric genes was similarly found to require KdmB. On the other hand, the COMPASS H3K4 methyltransferase complex subunit CclA that is required for H3K4 trimethylation in E. festucae represses the eas/ltm genes in culture conditions by maintaining H3K4me3 levels at the loci. Thus, these findings suggest a repressive role for H3K4me3 at these subtelomeric secondary metabolite loci and are consistent with the role of H3K4me3 in yeast telomeric silencing. Disruption of kdmB did not affect the symbiotic interaction of E. festucae with the host grass but severely reduced the levels of lolitrem B, an animal neurotoxin. At the same time, the levels of ergovaline, another animal toxin, and peramine, an insect feeding deterrent, were not affected. Therefore, disruption or inhibition of KdmB may also serve as a promising approach for future endophyte improvement programmes. The E. festucae homologue of KDM8 (an H3K36me2 demethylase), Jmj4, was further investigated in this study but no H3K6 demethylase activity was found for the protein. Both disruption and overexpression of the gene encoding Jmj4 similarly had no effect on the culture and symbiotic phenotypes of E. festucae. However, deletion of setB, encoding the homologue of yeast Set2 (H3K36 methyltransferase) specifically reduced histone H3K36me3 levels in E. festucae. This contrasts with deletion of Set2 in other fungi which affected H3K36 mono-, di- and trimethylation. The ΔsetB mutant was severely impeded in development, and was unable to establish infection of the host plant. Introduction of the wild-type setB gene reversed these phenotypes. This study shows that H3K4 trimethylation controlled by CclA and KdmB is an important regulator of subtelomeric secondary metabolite genes in E. festucae but not for the symbiotic interaction of the fungus with perennial ryegrass. On the other hand, the histone H3K36 methyltransferase SetB specifically controls H3K36 trimethylation in E. festucae and is required for normal vegetative growth and ability of the fungus to infect the host plant

Anti-insect defenses of Achnatherum robustum (sleepygrass) provided by two Epichloë endophyte species

Many pooid grasses (Poaceae) harbor Epichloë species (Hypocreales), endophytic fungi that often produce toxic alkaloids which may provide anti-insect protection for their hosts. Two natural populations of Achnatherum robustum (Vasey) (sleepygrass), in the Lincoln National Forest, Cloudcroft, and Weed (NM, USA), are infected with the endophyte species Epichloë funkii (KD Craven &amp; Schardl) JF White and Epichloë sp. nov. We tested whether: (1) these endophytes affect survival, growth, and development of the insect herbivore Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae) (fall armyworm), (2) larval diets alter adult fecundity (assessed as number of larvae or eggs produced by females and number of spermatophores that males transfer to females when enclosed in pairs within each feeding group), and (3) infections affect leaf consumption in larval no-choice and choice experiments. Individual larvae were reared on Epichloë infected vs. uninfected clipped leaves from the Cloudcroft and Weed population plants. Overall, armyworm survival was not affected when fed infected sleepygrass from either population. However, larvae that fed on Weed-infected plants were smaller and had longer development than larvae that fed on uninfected and Cloudcroft-infected plants. Males fed on Weed-infected leaves had reduced mating success. Interestingly, pupal mass increased when larvae fed on either the infected leaf types. However, heavier females from both infected diets did not lay more eggs than lighter females from uninfected diets. In a no-choice test, larvae on Weed-infected plants diet consumed more leaf biomass than larvae from three other groups. In choice tests, larvae avoided feeding on leaves infected with either of the endophytes relative to uninfected leaves. Thus, the two Epichloë may provide direct protection to sleepygrass from insect herbivory by deterrence. The Weed population endophyte may provide stronger indirect protection than the Cloudcroft endophyte by reducing insect fitness or increasing risks of predation and parasitism through delayed development, even though larvae may consume more leaf biomass

Disparate Independent Genetic Events Disrupt the Secondary Metabolism Gene \u3cem\u3eperA\u3c/em\u3e in Certain Symbiotic \u3cem\u3eEpichloë\u3c/em\u3e Species

Peramine is an insect-feeding deterrent produced by Epichloë species in symbiotic association with C3 grasses. The perA gene responsible for peramine synthesis encodes a two-module nonribosomal peptide synthetase. Alleles of perA are found in most Epichloë species; however, peramine is not produced by many perA-containing Epichloë isolates. The genetic basis of these peramine-negative chemotypes is often unknown. Using PCR and DNA sequencing, we analyzed the perA genes from 72 Epichloë isolates and identified causative mutations of perA null alleles. We found nonfunctional perA-ΔR* alleles, which contain a transposon-associated deletion of the perA region encoding the C-terminal reductase domain, are widespread within the Epichloë genus and represent a prevalent mutation found in nonhybrid species. Disparate phylogenies of adjacent A2 and T2 domains indicated that the deletion of the reductase domain (R*) likely occurred once and early in the evolution of the genus, and subsequently there have been several recombinations between those domains. A number of novel point, deletion, and insertion mutations responsible for abolishing peramine production in full-length perA alleles were also identified. The regions encoding the first and second adenylation domains (A1 and A2, respectively) were common sites for such mutations. Using this information, a method was developed to predict peramine chemotypes by combining PCR product size polymorphism analysis with sequencing of the perA adenylation domains