Epichloae endophytes of family Clavicipitaceae (comprising genera Epichloe and Neotyphodium) are fungal symbionts of Pooideae grasses. The associations formed, range from mutually beneficial to antagonistic and the nature of this relationship is dependent upon the importance of vertical (via host seeds) versus horizontal (ascospore mediated) transmission of the fungus. These endophytes can enhance their hosts’ survival through protection from abiotic and biotic stresses and can thus be utilized in an agricultural context. Animal-safe grass-endophyte associations that confer bio-protective properties for increased pasture persistence and productivity have been developed and commercialized. One of the crucial drivers underpinning the selection of epichloae strains for commercial development is endophyte derived bioactivity. The potential of next generation endophytes is determined by testing a number of attributes such as agronomic fitness, animal and food safety as well as compatibility with host plants of interest. Strategic research supports these activities by focusing on elucidating mechanisms of compatibility between host and fungal symbiont, as well as investigating other molecular drivers of symbiosis such as siderophore mediated iron-uptake, fungal signalling, fungal growth in host plants and fungal secondary metabolism. This review weaves together the different strands of multidisciplinary research aimed at ultimately exploiting epichloae endophytes for increased pasture performance.

The exploitation of epichloae endophytes for agricultural benefit

Summary
Plants form mutualistic symbioses with a variety of microorganisms including endophytic fungi that live inside the plant and cause no overt symptoms of infection. Some endophytic fungi form defensive mutualisms based on the production of bioactive metabolites that protect the plant from herbivores in exchange for a protected niche and nutrition from the host plant. Key elements of these symbioses are vertical transmission of the fungus through seed of the host plant, a narrow host range, and production of bioactive metabolites by the fungus.
Grasses frequently form symbioses with endophytic fungi belonging to the family Clavicipitaceae. These symbioses have been studied extensively because of their significant impacts on insect and mammalian herbivores. Many of the impacts are likely due to the production of four classes of bioactive alkaloids – ergot alkaloids, lolines, indole-diterpenes and peramine – that are distributed in different combinations among endophyte taxa.
Several legumes, including locoweeds, are associated with a toxic syndrome called locoism as a result of their accumulation of swainsonine. Species in two genera were recently found to contain previously undescribed endophytic fungi (Undifilum spp., family Pleosporaceae) that are the source of that toxin. The fungi are strictly vertically transmitted and have narrow host ranges.
Some plant species in the morning glory family (Convolvulaceae) also form symbioses with endophytic fungi of the Clavicipitaceae that produce ergot alkaloids and, perhaps in at least one case, lolines. Other species in this plant family form symbioses with undescribed fungi that produce swainsonine. The swainsonine-producing endophytes associated with the Convolvulaceae are distinct from the Undifilum spp. associated with locoweeds and the Clavicipitaceous fungi associated with Convolvulaceae.
In the establishment of vertically transmitted symbioses, fungi must have entered the symbiosis with traits that were immediately useful to the plant. Bioactive metabolites are likely candidates for such pre-adapted traits which were likely useful to the free-living fungi as well. With future research, vertically transmitted fungi from diverse clades with narrow host ranges and that produce bioactive compounds are likely to be found as important mutualists in additional plants.

https://besjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/1365-2435.12076

Bioactive alkaloids in vertically transmitted fungal endophytes

The epichloae: alkaloid diversity and roles in symbiosis with grasses

Chemical measures of bioavailability/bioaccessibility of PAHs in soil:fundamentals to application

The epichloae (Epichloe and Neotyphodium species), a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Pooideae). Most epichloae are vertically transmitted in seeds (endophytes), and most produce alkaloids that attack nervous systems of potential herbivores. These protective metabolites include ergot alkaloids and indole-diterpenes (tremorgens), which are active in vertebrate systems, and lolines and peramine, which are more specific against invertebrates. Several Epichloe species have been described which are sexual and capable of horizontal transmission, and most are vertically transmissible also. Asexual epichloae are mainly or exclusively vertically transmitted, and many are interspecific hybrids with genomic contributions from two or three ancestral Epichloe species. Here we employ genome-scale analyses to investigate the origins of biosynthesis gene clusters for ergot alkaloids (EAS), indole-diterpenes (IDT), and lolines (LOL) in 12 hybrid species. In each hybrid, the alkaloid-gene and housekeeping-gene relationships were congruent. Interestingly, hybrids frequently had alkaloid clusters that were rare in their sexual ancestors. Also, in those hybrids that had multiple EAS, IDT or LOL clusters, one cluster lacked some genes, usually for late pathway steps. Possible implications of these findings for the alkaloid profiles and endophyte ecology are discussed.

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Currencies of mutualisms: sources of alkaloid genes in vertically transmitted epichloae

Responses of adult Argentine stem weevil (ASW) to meadow fescue infected with the endophyte Neotyphodium uncinatum and to two loline alkaloids produced by the fungus were investigated Endophyte in two different meadow fescue seedlines did not affect adult ASW feeding scores but reduced oviposition compared with endophytefree controls In a nochoice experiment adult ASW were given artificial diets containing Nformyl loline (NFL) or Nacetyl norloline (NANL) at three concentrations or a control diet with no lolines Mortality was significantly higher at the highest rate of NFL than for lower rates and the control For NANL mortality was increased at both the lowest and the highest concentrations Feeding scores were higher on control than loline diets but only after 4 weeks In a choice experiment NFL appeared to have no effect on ASW feeding preference whereas there was less feeding at the highest concentration of NANL compared with the control diet

/pdf/argentine-stem-weevil-adults-are-affected-by-meadow-fescue-1sazbh07bk.pdf

Argentine stem weevil adults are affected by meadow fescue endophyte and its loline alkaloids

Orchard soils can contain elevated concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′-DDE), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p′-DDT), and heavy metals as a result of historical agrichemical applications. The bioavailability of p,p′-DDE, p,p′-DDT, As, Cd, Cu, and Pb from five field-aged New Zealand orchards and three grazing soils was assessed by using a 28-d bioassay with Aporrectodea caliginosa and chemical assays. Significant relationships were found between total soil and earthworm tissue concentrations for p,p′-DDE (p < 0.001), p,p′-DDT (p < 0.001), Cu (p < 0.001), and Pb (p < 0.01). Two neutral salt solutions, 0.01 M CaCl2 and 1 M NH4NO3, were used as surrogate measures of the bioavailability of heavy metals. Copper was the only heavy metal for which significant correlations were found between neutral-salt-extractable and earthworm tissue concentrations (p < 0.001). Up to 28% of the aged DDT residues were released from the soils by Tenax over a 24-h extraction period. Significant relationships (p < 0.01) between the Tenax-extractable and earthworm tissue concentrations for p,p′-DDE and p,p′-DDT showed that Tenax provides a good surrogate measure of the bioavailability of these compounds to A. caliginosa. Surprisingly, there was a similarly significant relationship (p < 0.001) between the total soil and earthworm tissue concentrations for p,p′-DDE and p,p′-DDT, suggesting that total soil concentrations alone were sufficient to predict uptake by A. caliginosa. These results demonstrate that the aged agrichemical residues in orchard soils, and particularly p,p′-DDE and p,p′-DDT, remain highly bioavailable to A. caliginosa despite decades of weathering and continue to present an environmental risk. Environ. Toxicol. Chem. 2012;31:1306–1316. © 2012 SETAC

Comparison of earthworm and chemical assays of the bioavailability of aged 1,1‐dichloro‐2,2‐bis(p‐chlorophenyl)ethylene, 1,1,1‐trichloro‐2,2‐bis(p‐chlorophenyl)ethane, and heavy metals in orchard soils

Epoxyjanthitrems I-IV (1-4) and epoxyjanthitriol (5) were isolated from seed of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Epichloe festucae var lolii Although structures for epoxyjanthitrems I-IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol (5) Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (Wiseana cervinata), a common pasture pest in New Zealand These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests

Identification and Structure Elucidation of Epoxyjanthitrems from Lolium perenne Infected with the Endophytic Fungus Epichloë festucae var. lolii and Determination of the Tremorgenic and Anti-Insect Activity of Epoxyjanthitrem I.

The root-feeding scarab insect Costelytra giveni causes severe damage to pasture ecosystems in New Zealand Loline alkaloids produced by some Epichloe endophytes deter this insect In two experiments, tall fescue infected with E coenophiala, strain AR584, and endophyte-free (Nil) controls were subjected to pulse drought stress (DS) or well-watered conditions (WW) The second experiment also included meadow fescue infected with E uncinata After 4-6 weeks exposure to the different conditions, roots were excised and fed to C giveni larvae for 7 days Relative root consumption (RC), frass production, and relative weight change (RWC) of larvae were measured and the loline content of roots determined RC and frass output were higher for larvae feeding on Nil DS tall fescue than WW and reduced by AR584 RWC was also greater on DS than on WW Nil plants but reduced by endophyte only in DS plants RC, frass output, and RWC of larvae were reduced by endophyte in DS and WW meadow fescue, but the effect was greater for WW plants Loline alkaloid concentration in roots was significantly higher in DS than WW tall fescue in Experiment I but higher in WW than DS meadow fescue in Experiment II These experiments have demonstrated that moisture status interacts with endophyte to differentially affect root herbivory in tall fescue and meadow fescue

https://www.mdpi.com/2076-2607/8/7/997/pdf

Root Herbivory: Grass Species, Epichloë Endophytes and Moisture Status Make a Difference.

Epichloe endophytes are filamentous fungi (family Clavicipitaceae) that live in symbiotic associations with grasses in the sub family Pooideae. In New Zealand, E. festucae var. lolii confers significant resistance to perennial ryegrass (Lolium perenne) against insect and animal herbivory and is an essential component of pastoral agriculture, where ryegrass is a major forage species. The fungus produces in planta a range of bioactive secondary metabolites, including ergovaline, which has demonstrated bioactivity against the important pasture pest black beetle, but can also cause mammalian toxicosis. We genetically modified E. festucae var. lolii strain AR5 to eliminate key enzymatic steps in the ergovaline pathway to determine if intermediate ergot alkaloid compounds can still provide insecticidal benefits in the absence of the toxic end product ergovaline. Four genes (dmaW, easG, cloA, and lpsB) spanning the pathway were deleted and each deletion mutant was inoculated into five different plant genotypes of perennial ryegrass, which were later harvested for a full chemical analysis of the ergot alkaloid compounds produced. These associations were also used in a black beetle feeding deterrence study. Deterrence was seen with just chanoclavine present, but was cumulative as more intermediate compounds in the pathway were made available. Ergovaline was not detected in any of the deletion associations, indicating that bioactivity towards black beetle can be obtained in the absence of this mammalian toxin.

Joanne G. Jensen

Papers

Argentine stem weevil adults are affected by meadow fescue endophyte and its loline alkaloids

Comparison of earthworm and chemical assays of the bioavailability of aged 1,1‐dichloro‐2,2‐bis(p‐chlorophenyl)ethylene, 1,1,1‐trichloro‐2,2‐bis(p‐chlorophenyl)ethane, and heavy metals in orchard soils

Identification and Structure Elucidation of Epoxyjanthitrems from Lolium perenne Infected with the Endophytic Fungus Epichloë festucae var. lolii and Determination of the Tremorgenic and Anti-Insect Activity of Epoxyjanthitrem I.

Root Herbivory: Grass Species, Epichloë Endophytes and Moisture Status Make a Difference.

Genetic Manipulation of the Ergot Alkaloid Pathway in Epichloë festucae var. lolii and Its Effect on Black Beetle Feeding Deterrence.