The growth promotion in endophyte symbiotic plants does not penalise the resistance to herbivores and bacterial microbiota.

A trade-off between growth and defence against biotic stresses is common in plants. Fungal endophytes of the genus Epichloë may relieve this trade-off in their host grasses since they can simultaneously induce plant growth and produce antiherbivore alkaloids that circumvent the need for host defence. The Epichloë ability to decouple the growth-defence trade-off was evaluated by subjecting ryegrass with and without Epichloë endophytes to an exogenous treatment with gibberellin (GA) followed by a challenge with Rhopalosiphum padi aphids. In agreement with the endophyte-mediated trade-off decoupling hypothesis, the GA-derived promotion of plant growth increased the susceptibility to aphids in endophyte-free plants but did not affect the insect resistance in endophyte-symbiotic plants. In line with the unaltered insect resistance, the GA treatment did not reduce the concentration of Epichloë-derived alkaloids. The Epichloë mycelial biomass was transiently increased by the GA treatment but at the expense of hyphal integrity. The response of the phyllosphere bacterial microbiota to both GA treatment and Epichloë was also evaluated. Only Epichloë, and not the GA treatment, altered the composition of the phyllosphere microbiota and the abundance of certain bacterial taxa. Our findings clearly demonstrate that Epichloë does indeed relieve the plant growth-defence trade-off.

Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

BACKGROUND: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. RESULTS: Expected relative increases in total fatty acids of 17-58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. CONCLUSIONS: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Can Aphid Herbivory Induce Intergenerational Effects of Endophyte-conferred Resistance in Grasses?

Plants have evolved mechanisms to survive herbivory. One such mechanism is the induction of defences upon attack that can operate intergenerationally. Cool-season grasses (sub-family Pooideae) obtain defences via symbiosis with vertically transmitted fungal endophytes (genus Epichloë) and can also show inducible responses. However, it is unknown whether these herbivore-induced responses can have intergenerational effects. We hypothesized that herbivory by aphids on maternal plants induces the intergenerational accumulation of endophyte-derived defensive alkaloids and resistance intensification in the progeny. We subjected mother plants symbiotic or not with Epichloë occultans, a species known for its production of anti-insect alkaloids known as lolines, to the aphid Rhopalosiphum padi. Then, we evaluated the progeny of these plants in terms of loline alkaloid concentration, resistance level (through herbivore performance), and shoot biomass. Herbivory on mother plants did not increase the concentration of lolines in seeds but it tended to affect loline concentration in progeny plants. There was an overall herbivore-induced intergenerational effect increasing the endophyte-conferred defence and resistance. Symbiotic plants were more resistant to aphids and had higher shoot biomass than their non-symbiotic counterparts. Since maternal herbivory did not affect the loline concentrations in seeds, the greater resistance of the progeny could have resulted from an inherited mechanism of epigenetic regulation. It would be interesting to elucidate the origin of this regulation since it could come from the host or the fungal symbiont. Thus, endophyte-driven differential fitness between symbiotic and non-symbiotic plants might be higher as generations pass on in presence of herbivores.

Ontogenetic and trans-generational dynamics of a vertically transmitted fungal symbiont in an annual host plant in ozone-polluted settings.

Tropospheric ozone is an abiotic stress of increasing importance in the context of global climate change. This greenhouse gas is a potent phytotoxic molecule with demonstrated negative effects on crop yield and natural ecosystems. Recently, oxidative stress has been proposed as a mechanism that could regulate the interaction between cool-season grasses and Epichloë endophytes. We hypothesized that exposure of Lolium multiflorum plants, hosting endophytes to an ozone-polluted environment at different ontogenetic phases, would impact the trans-generational dynamics of the vertically transmitted fungal symbiont. Here, we found that the ozone-induced stress on the mother plants did not affect the endophyte vertical transmission but it impaired the persistence of the fungus in the seed exposed to artificial ageing. Endophyte longevity in seed was reduced by exposure of the mother plant to ozone. Although ozone exposure did not influence either the endophyte mycelial concentration or their compound defences (loline alkaloids), a positive correlation was observed between host fitness and the concentration of endophyte-derived defence compounds. This suggests that fungal defences in grass seeds were not all produced in situ but remobilized from the vegetative tissues. Our study reveals ozone trans-generational effects on the persistence of a beneficial symbiont in a host grass.

Fungal Alkaloid Occurrence in Endophyte-Infected Perennial Ryegrass during Seedling Establishment.

The symbiotic Epichloë festucae var. lolii endophyte produces alkaloids which can provide its host grass, perennial ryegrass (Lolium perenne L), with a selective advantage in both natural and agricultural managed ecosystems. This study focuses on understanding the alkaloid concentrations that occur in endophyte-infected perennial ryegrass during the early establishment phase. In a glasshouse experiment fungal alkaloid concentrations (peramine, lolitrem B, ergovaline, and epoxy-janthitrems) were measured in perennial ryegrass seedlings infected with E. festucae var. lolii proprietary strains AR1, AR37, NEA2, and NZ common toxic for 69 days after sowing. The endophyte becomes metabolically active, starting alkaloid production, as early as 6 days after sowing. Alkaloid concentrations peaked in 8- to 10- day-old seedlings due to a seedling growth slowdown. This study provides data showing that the loss of insect protection in endophyte-infected seedlings is linked to a reduction in chemical defence after seed-stored, maternally synthesised alkaloids are diluted by seedling dry matter accumulation.

Correction: Fungal Alkaloid Occurrence in Endophyte-Infected Perennial Ryegrass during Seedling Establishment.

The original version of this article unfortunately contained a mistake. There is a mistake in the affiliation section and Figure 6. The correct affiliation and Figure 6 are shown here.

The plant hormone salicylic acid interacts with the mechanism of anti-herbivory conferred by fungal endophytes in grasses.

The plant hormone salicylic acid (SA) is recognized as an effective defence against biotrophic pathogens, but its role as regulator of beneficial plant symbionts has received little attention. We studied the relationship between the SA hormone and leaf fungal endophytes on herbivore defences in symbiotic grasses. We hypothesize that the SA exposure suppresses the endophyte reducing the fungal-produced alkaloids. Because of the role that alkaloids play in anti-herbivore defences, any reduction in their production should make host plants more susceptible to herbivores. Lolium multiflorum plants symbiotic and nonsymbiotic with the endophyte Epichloë occultans were exposed to SA followed by a challenge with the aphid Rhopalosiphum padi. We measured the level of plant resistance to aphids, and the defences conferred by endophytes and host plants. Symbiotic plants had lower concentrations of SA than did the nonsymbiotic counterparts. Consistent with our prediction, the hormonal treatment reduced the concentration of loline alkaloids (i.e., N-formyllolines and N-acetylnorlolines) and consequently decreased the endophyte-conferred resistance against aphids. Our study highlights the importance of the interaction between the plant immune system and endophytes for the stability of the defensive mutualism. Our results indicate that the SA plays a critical role in regulating the endophyte-conferred resistance against herbivores.

Complex Interactions among Sheep, Insects, Grass, and Fungi in a Simple New Zealand Grazing System.

Epichloë fungi (Ascomycota) live within aboveground tissues of grasses and can have important implications for natural and managed ecosystems through production of alkaloids. Nonetheless, vertebrate herbivores may possess traits, like oral secretions, that mitigate effects of alkaloids. We tested if sheep saliva mitigates effects of Epichloë alkaloids on a beetle pest of perennial ryegrass (Lolium perenne L.) in a New Zealand pasture setting. Plants with one of several fungal isolates were clipped with scissors, grazed by sheep, or clipped with sheep saliva applied to cut ends of stems. We then assessed feeding damage by Argentine stem weevils on blade segments collected from experimental plants. We found that clipping plants induced synthesis of an alkaloid that reduces feeding by beetles and that sheep saliva mitigates this effect. Unexpectedly, the alkaloid (perloline) that explains variation in beetle feeding is one produced not by the endophyte, but rather by the plant. Yet, these effects depended upon fungal isolate. Such indirect, complex interactions may be much more common in both managed and natural grassland systems than typically thought and could have implications for managing grazing systems.

Defining the pathways of symbiotic Epichloë colonization in grass embryos with confocal microscopy.

Asexual cool-season grass endophytes of the genus Epichloë (Ascomycota: Clavicipitaceae) are strictly vertically disseminated. The hosts of these mutualistic fungi express no symptoms during the fungal lifecycle that takes place entirely within the plant, while their hosts receive beneficial outcomes. These fungi are distributed in two major locations within the mature seeds of their hosts; namely, within the embryo (including the scutellum, coleoptile, plumule, radicle, and coleorhiza tissues) and between the aleurone and pericarp layers, with the latter hyphae playing no role in transmission of the fungus to the next plant generation. Conflicting evidence remains in the literature on the timing of embryo colonization. In a detailed investigation, utilizing confocal microscopy to observe the distribution of Epichloë coenophiala strain AR601 in tall fescue (Lolium arundinaceum), we tracked endophyte hyphal colonization in the ovary (pre-fertilization) through to the fully mature seed stage. Confocal microscopy images revealed that at the early and mature developmental stages of the embryo sac, before host grass fertilization, there were large quantities of endophyte mycelium present, especially around the antipodal cells, indicating that this endophyte enters the embryo sac before the fertilization stage. After host fertilization, fungal hyphae could be seen in the true embryo and early nonstarchy endosperm. Understanding the mechanisms of transmission to the seed is important for commercial seed producers and end users.

Translocation of Loline Alkaloids in Epichloë-Infected Cereal and Pasture Grasses: What the Insects Tell Us.

Aphids are major pests of cereal and pasture grasses throughout the world, vectoring disease and reducing plant production. There are few control options other than insecticides. Epichloë endophytes that produce loline alkaloids in their hosts provide a possible mechanism of control, with both meadow fescue and tall fescue naturally infected with loline-producing endophytes showing a resistance to Rhopalosiphum padi. We screened Elymus spp. naturally infected with endophytes that produced loline alkaloids at concentrations known to affect aphids on fescue but found no effect on these insects infesting Elymus. A synthetic loline-producing endophyte association with rye also had no effect on the aphids. After hypothesizing that the lolines were being translocated in the xylem in Elymus and rye rather than the phloem, we tested the rye and meadow fescue infected with loline-producing endophytes against a xylem feeding spittlebug. The endophyte in rye inhibited the feeding of the insect and reduced its survival, whereas the endophyte-infected meadow fescue had no effect on the spittlebug but reduced the number of aphids. Lolines applied to the potting medium of endophyte-free and endophyte-infected rye, ryegrass, and tall fescue resulted in a decrease in the aphid populations on the endophyte-free pasture grasses relative to the untreated controls but had no effect on aphid numbers on the rye. We tentatively conclude that lolines, produced in both natural and synthetic association with Elymus and rye, are partitioned in the xylem rather than the phloem, where they are inaccessible to aphids.

Characterization of the Biosynthetic Gene Cluster for the Ribosomally Synthesized Cyclic Peptide Epichloëcyclins in Epichloë festucae.

The various grass-induced epichloëcyclins of the Epichloë spp. are ribosomally synthesized and post-translationally modified peptides (RiPPs), produced as small, secreted cyclopeptides from a single gene, gigA. Here, four clustered and coregulated genes (gigA, gigB, gigC, and kexB) with predicted roles in epichloëcyclin production in Epichloë festucae were evaluated through gene disruption. Subsequent chemical analysis indicates that GigB is a DUF3328 domain-containing protein associated with cyclization of epichloëcyclins; GigC is a methyltransferase enzyme responsible for N-methylation of desmethylepichloëcyclins; and KexB is a subtilisin-like enzyme, partly responsible for the propeptide cleavage of epichloëcyclin intermediates. Symbiotic effects on the host phenotype were not observed for gigA, gigC, or kexB mutants, although ΔgigB infection correlated with increased host tiller height and biomass, while only ΔkexB exhibited an effect on endophyte morphology. Disrupting epichloëcyclin biosynthesis showed negligible influence on the biosynthesis of E. festucae-associated alkaloids. Epichloëcyclins may perform other secondary metabolism functions in Epichloë and other fungi.

Identification of Genetic Diversity, Pyrrocidine-Producing Strains and Transmission Modes of Endophytic Sarocladium zeae Fungi from Zea Crops.

Genotyping by sequencing (GBS) was used to reveal the inherent genetic variation within the haploid fungi Sarocladium zeae isolated from diverse Zea germplasm, including modern Zea mays and its wild progenitors-the teosintes. In accordance with broad host relationship parameters, GBS analysis revealed significant host lineages of S. zeae genetic diversity, indicating that S. zeae genetic variation may associate with different evolutionary histories of host species or varieties. Based on a recently identified PKS-NRPS gene responsible for pyrrocidine biosynthesis in S. zeae fungi, a novel PCR assay was developed to discriminate pyrrocidine-producing S. zeae strains. This molecular method for screening bioactive strains of S. zeae is complementary to other approaches, such as chemical analyses. An eGFP-labelled S. zeae strain was also developed to investigate the endophytic transmission of S. zeae in Z. mays seedlings, which has further improved our understanding of the transmission modes of S. zeae endophytes in maize tissues.

Dissection of the epoxyjanthitrem pathway in Epichloë sp. LpTG-3 strain AR37 by CRISPR gene editing.

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.

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

The root-feeding scarab insect Costelytra giveni causes severe damage to pasture ecosystems in New Zealand. Loline alkaloids produced by some Epichloë 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.

The Impact of Endophyte Infection, Seed Aging, and Imbibition on Selected Sugar Metabolite Concentrations in Seed.

This study investigated effects of seed aging and imbibition on sugar metabolite concentrations in Epichloë endophyte-infected and endophyte-free seed of tall fescue ( Festuca arundinacea Schreb.). Two treatments, namely, accelerated aging and imbibition, were applied to the seeds, with embryo and endosperm tissues analyzed separately. Gas chromatography with flame ionization detection was employed for analysis of sugar metabolites within the seed tissues. Mannitol, ribitol, and trehalose were more abundant in embryo than endosperm tissues and were identified at consistently higher concentrations within endophyte-infected compared to endophyte-free seeds. The ratio of raffinose to sucrose significantly increased with seed aging in both endophyte-free and endophyte-infected embryo tissues, while significantly lower concentrations of trehalose were detected in tissues dissected from aged-seed regardless of endophyte status. This research provides fundamental insight into the metabolic details of endophyte survival in seed and provides a first evaluation of key carbohydrates present in the fungal-plant symbiosis.

Sipha maydis sensitivity to defences of Lolium multiflorum and its endophytic fungus Epichloë occultans.

BACKGROUND: Plants possess a sophisticated immune system to defend from herbivores. These defence responses are regulated by plant hormones including salicylic acid (SA) and jasmonic acid (JA). Sometimes, plant defences can be complemented by the presence of symbiotic microorganisms. A remarkable example of this are grasses establishing symbiotic associations with Epichloë fungal endophytes. We studied the level of resistance provided by the grass' defence hormones, and that provided by Epichloë fungal endophytes, against an introduced herbivore aphid. These fungi protect their hosts against herbivores by producing bioactive alkaloids. We hypothesized that either the presence of fungal endophytes or the induction of the plant salicylic acid (SA) defence pathway would enhance the level of resistance of the grass to the aphid. METHODS: Lolium multiflorum plants, with and without the fungal endophyte Epichloë occultans, were subjected to an exogenous application of SA followed by a challenge with the aphid, Sipha maydis. RESULTS: Our results indicate that neither the presence of E. occultans nor the induction of the plant's SA pathway regulate S. maydis populations. However, endophyte-symbiotic plants may have been more tolerant to the aphid feeding because these plants produced more aboveground biomass. We suggest that this insect insensitivity could be explained by a combination between the ineffectiveness of the specific alkaloids produced by E. occultans in controlling S. maydis aphids and the capacity of this herbivore to deal with hormone-dependent defences of L. multiflorum.

Variation in Alkaloid Production from Genetically Diverse Lolium Accessions Infected with Epichloë Species.

Widespread infection of Epichloë occultans in annual ryegrass in Australia suggests that infection provides its weedy host, Lolium rigidum, some ecological advantage. Initial studies determined the distribution and profiles of known Epichloë alkaloids (epoxy-janthitrems, ergovaline, lolines, lolitrem B, and peramine) in plant extracts using a combination of GC-FID and HPLC techniques utilizing a single accession of Australian L. rigidum. However, the lolines N-acetylnorloline (NANL) and N-formylloline (NFL) were the only alkaloids detected and were highly concentrated in the immature inflorescences of mature plants. Additional glasshouse studies subjected a wide range of Australian L. rigidum haplotypes and international annual Lolium accessions to a suite of analyses to determine alkaloid levels and profiles. Again, NFL and NANL were the key lolines produced, with NFL consistently predominating. Considerable variation in alkaloid production was found both within and between biotypes and accessions evaluated under identical conditions, at the same maturation stage and on the same tissue type. The pyrrolopyrazine alkaloid peramine was also present in 8 out of 17 Australian biotypes of L. rigidum and 7 out of 33 international accessions infected with Epichloë spp.; the highest peramine concentrations were observed in seed extracts from L. rigidum collected from Australia. This study represents the first report of alkaloids from a geographically diverse collection of annual ryegrass germplasm infected with Epichloë spp. when grown under identical controlled conditions.

Variation in the expression of ergot alkaloids between individual tillers of perennial ryegrass.

Epichloë fungal endophytes of cool season grasses are well-known to produce a range of alkaloids of benefit to the host. Some of these compounds are advantageous to agriculture due to qualities that promote pasture persistence (e.g., the loline class of alkaloids confer insect protection) while others are detrimental to the well-being of grazing livestock. The ergot alkaloids (e.g., ergovaline), produced in ryegrass and tall fescue associations, causes poor animal health in farming regions in many countries around the world and further study is required to improve our knowledge on this class of compounds. Here we present the application of a quantitative LC-MS/MS (liquid chromatography coupled to mass spectrometry) method measuring eight ergot alkaloids (chanoclavine, agroclavine, elymoclavine, lysergol, lysergic acid, ergine, lysergyl-alanine, ergovaline) produced by endophyte infected grasses, to monitor levels in individual tillers from multiple plants of a single cultivar of perennial ryegrass (Lolium perenne cv. "Grasslands Samson") infected with a common toxic endophyte strain (Epichloë festucae var. lolii). Monitoring the expression in individual tillers allows an estimation of the variability within a plant (between tillers) as well as between plants. The study showed that there is significant variation in the concentration of the ergot alkaloids between tillers of a single plant, at or exceeding the level of variation observed between individual plants of a population. This result emphasizes the fundamental importance of robust experimental design and sampling procedures when alkaloid expression assessment is required and these need to be rigorously tailored to the hypothesis being tested.

Endophyte-mediated resistance to black cutworm as a function of plant cultivar and endophyte strain in tall fescue.

To improve Neotyphodium endophyte-mediated resistance to black cutworm Agrotis ipsilon (Hufnagel) (BCW), a series of experiments was conducted by using several different cultivars of tall fescue, Schedonorus arundinaceus (Schreb.) Dumort. in combination with several different haplotypes of the endophyte Neotyphodium coenophialum (Morgan-Jones & Gams) (plant cultivar × endophyte haplotype = plant line), each producing unique alkaloid profiles. BCW settling response, survival at 5 and 10 d, and larval biomass varied significantly among plant lines. In general, greater variation BCW performance was observed within a single plant cultivar infected with different endophyte haplotypes than among different plant cultivars infected with the same endophyte haplotype, but comparisons among the former were far more numerous. Although five endophyte-mediated alkaloids representing three alkaloid classes were quantified in the plants, the pyrrolizidine alkaloid N-acetyl norloline was consistently the single best predictor of BCW performance. BCW settling response, 5-d survival, and 10-d survival decreased as levels of the alkaloid N-acetyl norloline increased. The same three response variables also decreased with increasing levels of peramine, but increased with increasing levels of ergovaline. Minor variation in endophyte infection levels occurring among infected plant lines had no significant influence on BCW performance. Results indicate a potentially important role for N-acetyl norloline and peramine in providing resistance to black cutworm whereas ergovaline appears to be much less important. Therefore, endophyte haplotypes expressing high levels of N-acetyl norloline and peramine may be of particular importance for developing 'friendly' endophyte-enhanced turf and pasture grasses that resist challenging lepidopteran pests, although remaining safe for wildlife and grazing mammals.

Potentiometric evaluation of calix[4]arene anion receptors in membrane electrodes: phosphate detection.

Ion-selective membrane electrodes doped with the urea- or thiourea-functionalised calix[4]arenes, 5,11,17,23-tetra-tert-butyl-25,27-bis[[4-N'-(phenylureido)butyl]oxy]-26,28-dipropoxy calix[4]arene (I) and 5,11,17,23-tetra-tert-butyl-25,27-bis[[4-(N'-phenylthioureido)-butyl]oxy]-26,28-dipropoxy calix[4]arene (II), were evaluated for anion sensing. Potentiometric results show that these calixarene ionophore-based membrane electrodes exhibit a good sensitivity to aqueous solutions of the monohydrogen orthophosphate species HPO(4)(2-) in the concentration range 5.0 x 10(-5) to 1.0 x 10(-1)M, with near-Nernstian response slopes of -33.0 and -28.0 mV dec(-1) for ionophores I and II, respectively. Selectivity coefficient values for monohydrogen orthophosphate over a range of common anions were determined by the fixed interference and matched potential methods and indicated that these membrane electrodes exhibit a good selectivity for HPO(4)(2-) with respect to the other anions, including sulfate and nitrate.