Distribution of NRPS gene families within the Neotyphodium/Epichloë complex

Neotyphodium and Epichloë spp are closely related asexual and sexual endophytic fungi, respectively, that form mutualistic associations with cool season grasses of the subfamily Pooideae. The endophytes confer a number of advantages to their hosts, but also can cause animal toxicoses and these effects are, in many cases, due to the production of fungal secondary metabolites. In filamentous fungi, secondary metabolite genes are commonly clustered and, for those pathways involved in non-ribosomal peptide synthesis, a non-ribosomal peptide synthetase (NRPS) gene is always found as a key component of the cluster. Members of this gene family encode large multifunctional enzymes that synthesize a diverse range of bioactive compounds and in numerous cases have been shown to serve as pathogenicity or virulence factors, in addition to suggested roles in niche adaptation. We have used a degenerate PCR approach to identify members of the NRPS gene family from symbiotic fungi of the Neotyphodium/Epichloë complex, and have shown that collectively, at least 12 NRPS genes exist within the genomes examined. This suggests that secondary metabolites are important during the life cycles of these fungi with their hosts. Indeed, both the ergovaline and peramine biosynthetic pathways, which confer competitive abilities to Neotyphodium and Epichloë symbioses, contain NRPS genes at their core. The distribution of these genes among different Neotyphodium/Epichloë lineages suggests that a common ancestor contributed most of the complement of NRPS genes, which have been either retained or lost during the evolution of these fungi.     

Disparate Independent Genetic Events Disrupt the Secondary Metabolism Gene perA in Certain Symbiotic Epichlo? 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.     

Endoplasmic reticulum localized PerA is required for cell wall integrity,azole drug resistance,and virulence in Aspergillus fumigatus

GPI‐anchoring is a universal and critical post‐translational protein modification in eukaryotes. In fungi, many cell wall proteins are GPI‐anchored, and disruption of GPI‐anchored proteins impairs cell wall integrity. After being synthesized and attached to target proteins, GPI anchors undergo modification on lipid moieties. In spite of its importance for GPI‐anchored protein functions, our current knowledge of GPI lipid remodelling in pathogenic fungi is limited. In this study, we characterized the role of a putative GPI lipid remodelling protein, designated PerA, in the human pathogenic fungus Aspergillus fumigatus. PerA localizes to the endoplasmic reticulum and loss of PerA leads to striking defects in cell wall integrity. A perA null mutant has decreased conidia production, increased susceptibility to triazole antifungal drugs, and is avirulent in a murine model of invasive pulmonary aspergillosis. Interestingly, loss of PerA increases exposure of β‐glucan and chitin content on the hyphal cell surface, but diminished TNF production by bone marrow‐derived macrophages relative to wild type. Given the structural specificity of fungal GPI‐anchors, which is different from humans, understanding GPI lipid remodelling and PerA function in A. fumigatus is a promising research direction to uncover a new fungal specific antifungal drug target.     

Botanophila flies on Epichloë host species in Europe and North America: no evidence for co-evolution

Species of Epichloë (Ascomycota: Clavicipitaceae), which infect grasses, maintain an obligate symbiotic relationship with flies of the genus Botanophila (Diptera: Anthomyiidae). Sexual reproduction of the fungus usually requires a visit by Botanophila, which serves to ‘pollinate’ the fungus with spermatia of the opposite mating type; the flies in turn deposit their eggs on fungal tissues upon which the larvae feed. For a molecular phylogenetic study, a total of 108 fly larvae were collected from 10 different Epichloë species on various grasses in Europe and North America. Sequence analysis of the mitochondrial cytochrome oxidase gene (COII) detected six distinct Botanophila taxa that were associated with Epichloë. Three were restricted to samples from Europe, two to samples from North America, and one was present on both continents. In North America the common taxon [identified as Botanophila lobata (Collin)] was found on Epichloë hosts of native grasses and on Epichloë typhina (Pers.: Fr.) Tul. and Epichloë festucae Leuchtm., Schardl and M.R. Siegel of the introduced grasses Dactylis glomerata L. and Festuca rubra L., respectively. In a maximum likelihood phylogram, one of the American taxa was most basal, while the other was nested within the European taxa, suggesting that European and American taxa of Epichloë-associated Botanophila spp. may not have separate origins. Analyses of the fly populations further indicated that up to four different Botanophila taxa can be present at a single location. On taxon level there was no specificity of flies for Epichloë host species (except taxon 6 that was found only on Epichloë glyceriae Schardl & Leuchtmann), although several taxa appeared to prefer some hosts over others. Comparison of molecular phylogenetic relationships of Botanophila species with those of the associated Epichloë hosts did not suggest co-evolution of the fungus and the fly. Thus, associations between Botanophila flies and Epichloë hosts may have arisen independently more than once.     

Efficacy of an oil-based formulation combining Metarhizium rileyi and nucleopolyhedroviruses against lepidopteran pests of soybean

A biopesticide that combines Metarhizium rileyi and nucleopolyhedrovirus (NPV) in an oil dispersion (OD) was developed. Its efficacy against Anticarsia gemmatalis and Chrysodeixis includens was evaluated under laboratory and field conditions. First, each of the two selected fungal strains, combined or not in OD preparations with AgMNPV or ChinNPV, was tested in the laboratory against A. gemmatalis and C. includens, respectively. AgMNPV–fungus combinations had similar effect on overall mortality of A. gemmatalis compared to each pathogen used alone. C. includens was less susceptible to infection by M. rileyi strains, and ChinNPV became the major cause of larval death. The OD formulations with M. rileyi and/or NPV were applied in infested soybean fields. ChinNPV + M. rileyi caused 50.8% of overall mortality six days after spraying in a soybean crop infested by both pests, whereas for treatments with the fungus or the virus alone the percentages were 41.2% and 8.8%, respectively. In another field infested solely by A. gemmatalis, insect mortality was similar for the AgMNPV–fungus combination and the virus used alone. Larval parasitism by wasps and dipterans was not affected following biopesticide spraying. Our results suggest that the performance of a dual-action biopesticide is related to the NPV and fungal strains that are combined and the proportion of host populations simultaneously infesting the crop.     

Metarhizium (Nomuraea) rileyi as biological control agent

Metarhizium rileyi is an entomopathogenic fungus of Lepidoptera and infects many noctuids, which are key pests of agricultural crops. In this literature review, we summarise the state of art and use of this potential biological pest control agent. Pathogen–host interactions include conidial adhesion, invasion process, and enzymatic activity. Environmental factors that impact the productivity associated with this fungus such as environment temperature, humidity, and availability of spores are discussed. Recent research has focused on enzymatic aspects related to pathogenicity, in which the cause is not fully elucidated yet. More than 60 known host species, mainly lepidopterans, are listed. Non-susceptible species, including predatory insects, are also presented. The difficulty of mass production and the formulation are the principal obstacles in use of M. rileyi. We summarise the challenges necessary to better utilise this agent in biocontrol and suggest novel research approaches, including the development of commercial and technically viable formulations.     

Manipulation of host ecdysteroid hormone levels facilitates infection by the fungal insect pathogen,Metarhizium rileyi

Entomopathogenic fungi such as Metarhizium rileyi and Beauveria bassiana are widely used insect biological control agents. Little, however, is known concerning genetic or enzymatic factors that differentiate the mechanisms employed by these two fungal pathogens to infect target hosts. Infection by either of these organisms is known to increase levels of the growth and molting hormone, ecdysone, which also regulates the expression of a number of innate immune pathways. M. rileyi, but not B. bassiana, has apparently evolved an ecdysteroid-22-oxidase (MrE22O) that inactivate ecdysone. We show that deletion of MrE22O impaired virulence compared with the wild-type strain, with an increase in ecdysone titer seen in hosts that was coupled to an increase in the expression of antimicrobial genes. An M. rileyi strain engineered to overexpress MrE22O (MrE22O^OE), as well as trans-expression in B. bassiana (Bb::MrE220^OE) resulted, in strains displaying enhanced virulence and dampening of host immune responses compared with their respective wild-type parental strains. These results indicate that ecdysone plays an important role in mediating responses to fungal infection and that some insect pathogenic fungi have evolved mechanisms for targeting this hormone as a means for facilitating infection.     

Creating and screening Cochliobolus heterostrophus non-ribosomal peptide synthetase mutants

An exhaustive characterization of the set of non-ribosomal peptide synthetase (NRPS) genes of the corn pathogen, Cochliobolus heterostrophus, and the small molecule peptides produced by the enzymes they encode, has been undertaken to ascertain the role of the peptide metabolites in the fungal cell. To date, the NRPS method of peptide biosynthesis has been described for filamentous ascomycete fungi (and to a limited extent, for basidiomycete fungi) and for bacteria, only. In addition to structural diversity, non-ribosomal peptides have a broad spectrum of biological activities, many are useful in medicine, agriculture, industry, and biological research. However, to suggest that inter-organismal activities is their primary function is likely incorrect; in fact, the physiological significance of these peptides to the producing fungi is largely unknown. We document that NRPS enzymes are purveyors of small molecules for both basal metabolism and for specialized environmental niches and that some are conserved, but most are not.     

Sympatric Epichloë species and chemotypic profiles in natural populations of Lolium perenne

The distribution of different Epichloë species within eight natural populations of Lolium perenne was studied. In total, 40.2% of the asymptomatic plants were infected by Epichloë festucae var. lolii or by Epichloë typhina. Both species occurred in sympatry in seven grass populations, and some plants had dual infections by both taxa. No hybrid taxa such as Epichloë hybrida were detected. Epichloë festucae strains were classified into two morphotypes, M1 and M3, according to culture characters, both morphotypes occurred in sympatry in seven populations. Plants bearing stromata produced by Epichloë typhina were observed, but asymptomatic plants infected by this species also occurred in seven populations. The alkaloid profile of Lolium perenne plants was related to the morphotype of their infecting strains: most plants infected by M3-strains were characterized by lolitrem, and those with M1-strains contained either ergovaline or lolitrem. Plants infected by Epichloë typhina were characterized by high peramine content.     

Chemotypic diversity of epichloae,fungal symbionts of grasses

The epichloid fungi – comprising sexual Epichloë species and asexual Neotyphodium species – are symbionts of cool-season grasses (subfamily Poöideae), mostly vertically transmissible (seedborne), and well known for production of anti-herbivore alkaloids. Four classes of alkaloids are known to be produced by epichloae: lolines (saturated aminopyrrolizidines), indole–diterpenes, ergot alkaloids, and peramine. There is a wide range of chemotypic diversity among and even within epichloid species. At the molecular level, this diversity may in part reflect the telomeric association of two of the four alkaloid biosynthesis gene clusters. Ecologically, the chemotypic diversity within species may reflect frequency-dependent selection for the alkaloids, which provide defences against insects and, in some cases, vertebrates, but can be expensive to produce. Interspecific hybridization, common among asexual epichloae, can pyramid the alkaloid biosynthesis genes. Compared to sexual epichloae, many asexual epichloae produce high levels of alkaloids – particularly lolines – suggesting that strict vertical transmission selects for enhanced capability of host protection.     

Peramine and other fungal alkaloids are exuded in the guttation fluid of endophyte-infected grasses

Many grasses live in association with asymptomatic fungi (Neotyphodium spp. endophytes), which grow in the intercellular spaces of the grass. These endophytes produce a range of alkaloids that protect the grass against grazing by mammals and insects. One of these alkaloids is an unusual pyrrolopyrazine, peramine. Peramine appears to be continuously produced by the endophyte, but does not progressively accumulate. No mechanism for the removal of peramine by its further metabolism or any other process has been reported. Our aim was to detect peramine or peramine metabolites in plant fluids to determine if peramine is mobilized, metabolized or excreted by the plant. We also wanted to determine if other fungal metabolites are mobilized by the plant, as has been proposed for the loline alkaloids.We developed a highly sensitive method for the analysis of peramine, using a linear ion trap mass spectrometer. We studied the fragmentation pathway of peramine using ESI MSⁿ and ESI FTICRMS. Based on these results we developed a single reaction monitoring method using the fragmentation of the guanidinium moiety. Cut leaf fluid and guttation fluid of different grass endophyte associations (Lolium perenne with Neotyphodium lolii, Festuca arundinacea with Neotyphodium coenophialum, and Elymus sp. with Epichloë sp.) were analysed. Peramine was detected in the cut leaf fluid of all grass-endophyte associations, but not in the guttation fluid of all associations. In some associations we also detected lolines and ergot peptide alkaloids. This is the first report showing the mobilization of fungal alkaloids into plant fluids by the host plant in grass-endophyte associations.     

The Botanophila–Epichloë association in cultivated Festuca in Oregon: evidence of simple fungivory

We investigated the Botanophila (Diptera: Anthomyiidae)–Epichloë (Ascomycetes: Clavicipitaceae) interaction in cultivated Festuca spp. (fine fescue) in Oregon in western USA. Epichloë spp. are endophytic fungi of grasses in the subfamily Pooideae. They develop a felt‐like stroma on the surface of grass culms and a dense mycelium within the culms that typically prevents seed head emergence. As a result, seed yields are suppressed, and hence the disease is known as choke. Studies of Epichloë spp. on wild grasses indicate that the fly–fungus interaction is an obligatory mutualism. During oviposition, Botanophila transfers Epichloë spermatia between stromata of opposite mating types, and the perithecia that develop after fungal fertilization serve as food for Botanophila larvae. In the current study, we surveyed 19 cultivated fields of Festuca spp. in Oregon, and observed choke caused by Epichloë festucae Leuchtmann, Schardl and Siegl in 10 of these. However, perithecia were observed in only four fields, and on only 1.0–2.6% of stromata. Perithecial development was also low, and rarely covered 50% of the stroma surface. Despite the absence or low frequency of fertilized stromata, Botanophila lobata Collin larvae were present in all choke‐infested fields. Infestation levels ranged from 2.5 to 70.7%, based on an examination of 175–200 stromata from each field. Only eight (= 2%) of the 450 stromata with B. lobata had perithecia, and the greater majority of B. lobata larvae completed development and exited from unfertilized stromata. This is the first report of the B. lobata–E. festucae association in the USA, and of B. lobata larvae developing successfully on unfertilized Epichloë stromata. The average pupal weight (0.0032 g) did not differ significantly from pupae (0.0030 g) originating from larvae that had developed on fertilized stromata of E. typhina on Dactylis glomerata in a neighboring field. This result indicates that in cultivated fine fescue fields in Oregon, B. lobata forages on E. festucae, but fly development is not dependent on the fertilized stromata of Epichloë.     

Can Epichloë endophytes enhance direct and indirect plant defence?

Induced or constitutive production of secondary metabolites is a successful plant defence strategy against herbivores which can be mediated by plant associated micro-organisms. Several grass species can be associated with an endophytic fungus of the genus Epichloë which produces herbivore toxic or deterring alkaloids. Besides these direct defences, herbivorous insects are controlled via indirect plant defence mechanisms by attracting predators. Recent studies indicate that Epichloë endophytes can improve the grass emitted volatile organic compounds towards herbivore deterrence. Due to their defensive mutualistic function, we hypothesize that Epichloë altered plant volatiles can attract aphid predators and contribute to an increased indirect plant defence. With a common garden study, we show that hoverfly (Syrphidae) larvae and pupae were more abundant on endophyte-infected plants compared to uninfected plants. Our results indicate that the Epichloë endophyte provides, besides direct defence (alkaloid), indirect plant defence by improving the plant odor attracting more olfactory foraging aphid predators. Future research is needed in order to understand: (I) whether endophyte-mediated changes in plant volatiles are induced herbivore specific, (II) whether there is a trade-off between endophyte-mediated direct and indirect plant defence, (III) whether the endophyte produces volatiles or induces a change in plant-derived volatiles, (IV) the role of plant signals in endophyte-mediated plant defence.     

Identification of extracellular siderophores and a related peptide from the endophytic fungus Epichloë festucae in culture and endophyte-infected Lolium perenne

A number of genes encoding non-ribosomal peptide synthetases (NRPSs) have been identified in fungi of Epichloë/Neotyphodium species, endophytes of Pooid grasses, including sidN, putatively encoding a ferrichrome siderophore-synthesizing NRPS. Targeted gene replacement and complementation of sidN in Epichloë festucae has established that extracellular siderophore epichloënin A is the major product of the SidN enzyme complex (Johnson et al., 2007a). We report here high resolution mass spectrometric fragmentation experiments and NMR analysis of an isolated fraction establishing that epichloënin A is a siderophore of the ferrichrome family, comprising a cyclic sequence of four glycines, a glutamine and three N^δ-trans-anhydromevalonyl–N^δ-hydroxyornithine (AMHO) moieties. Epichloënin A is unusual among ferrichrome siderophores in comprising an octapeptide rather than hexapeptide sequence, and in incorporating a glutamine residue. During this investigation we have established that desferrichrome siderophores with pendant trans-AMHO groups can be distinguished from those with pendant cis-AMHO groups by the characteristic neutral loss of an hydroxyornithine moiety in the MS/MS spectrum. A minor component, epichloënin B, has been characterized as the triglycine variant by mass spectrometry. A peptide characterized by mass spectrometry as the putative deoxygenation product, epichloëamide has been detected together with ferriepichloënin A in guttation fluid from ryegrass (Lolium perenne) plants infected with wild-type E. festucae, but not in plants infected with the ΔsidN mutant strain, and also detected at trace levels in wild-type E. festucae fungal culture.     

The interaction of a Botanophila fly species with an exotic Epichloë fungus in a cultivated grass: fungivore or mutualist?

Epichloë spp. (Ascomycetes: Clavicipitaceae) are endophytic fungii of Pooid grasses that cause choke disease, the suppression of seed production. They also host Botanophila spp. (Diptera: Anthomyiidae), the larvae of which feed on the fungus. Studies on Epichloë elymi on wild grasses indicate that the flies transfer spermatia between Epichloë mating types, thereby affecting cross‐fertilization, suggesting that the fungus–fly interaction reflects obligatory mutualism. Epichloë typhina, inadvertently introduced into western USA, was first detected in cultivated Dactylis glomerata L. fields in 1996. It spread rapidly, raising concerns about impacts on seed production. The present study was conducted to address questions pertaining to the occurrence and nature of the fungus–fly interaction in the new habitat of E. typhina. The first report of an endemic Botanophila species associated with E. typhina in Oregon is presented here. Surveys of D. glomerata fields indicated no correlation between fly abundance and fungal fertilization. In one field, no fly eggs or larvae were detected, but fertilized stromata were universally present. The fly was established in the remaining 12 fields surveyed, but while the number of stromata with fly larvae ranged from 6 to 98%, stromata development was uniform. Up to 10 larvae were present on a stroma, and these consumed >90% of the perithecia. Comparisons of pupal weights indicated that the fungal resource was not limiting, even at high larval densities. An exclusion study in a D. glomerata field also indicated that E. typhina fertilization occurred without the fly. In Oregon, the fly clearly benefits from the association with the fungus, but there is no evidence of benefit to the fungus. Thus if obligatory mutualism in the fungus–fly relationship described from the midwestern USA is the norm, our studies suggest a shift in the interaction to one of simple foraging on the fungus by fly larvae.     

A technique for accelerating and synchronising germination of conidia of the entomopathogenic fungus Metarhizium anisopliae

The entomopathogenic fungus Metarhizium anisopliae (strain ME1) failed to swell or form germ-tubes in distilled water. However, a period of soaking in distilled water (10–44 h) accelerated the process of germination when a suitable nutrient source was provided. The implications of this novel observation are discussed in terms of mechanisms of germination and the use of parasitic fungi for insect pest control.