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.

Factors controlling the effects of mutualistic bacteria on plants associated with fungi.

Plants interacting with mutualistic fungi (MF) or antagonistic fungi (AF) can form associations with bacteria. We assessed whether the performance gain conferred by mutualistic bacteria to fungal-associated plants is affected by the interaction between symbiont traits, type of bacterial-protective traits against AF and abiotic/biotic stresses. Results showed that (A) performance gain conferred by bacteria to MF-associated plants was greater when symbionts promoted distinct rather than similar plant functions, (B) bacterial-based alleviation of the AF's negative effect on plants was independent of the type of protective trait, (C) bacteria promoted a greater performance of symbiotic plants in presence of biotic, but not abiotic, stress compared to stress-free situations. The plant performance gain was not affected by any fungal-bacterial trait combination but optimised when bacteria conferred resistance traits in biotic stress situations. The effects of bacteria on fungal-associated plants were controlled by the interaction between the symbionts' functional traits and the relationship between bacterial traits and abiotic/biotic stresses.

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.

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.

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.

Assessment of fluorescein-based fluorescent dyes for tracing Neotyphodium endophytes in planta.

Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea, respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxy-fluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor while M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fungal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fluoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed.

Survey of the Endophytic Bacteria Inhabiting Wild Daucus Seed Using 16S rRNA Gene Amplicon Sequencing.

We report a preliminary survey of the endophytic bacterial microbiota of seed from wild carrot (Daucus carota) using 16S rRNA gene amplicon sequencing. Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were the most abundant phyla detected, while Bacillus, Massilia, Paenibacillus, Pantoea, Pseudomonas, Rhizobium, Sphingomonas, and Xanthomonas were the most abundant genera.

Biological Control of Diamondback Moth-Increased Efficacy with Mixtures of Beauveria Fungi.

Diamondback moth (DBM) is an important horticultural pest worldwide as the larvae of these moths feed on the leaves of cruciferous vegetables. As DBM has developed resistance to more than 100 classes of synthetic insecticides, new biological control options are urgently required. Beauveria species are entomopathogenic fungi recognized as the most important fungal genus for controlling a wide range of agricultural, forestry, and veterinary arthropod pests. Previous research, aimed at developing new Beauveria-based biopesticides for DBM, has focused on screening single isolates of Beauveria bassiana. However, these fungal isolates have individual requirements, which may limit their effectiveness in some environments. This current study separately assessed 14 Beauveria isolates, from a range of habitats and aligned to four different species (Beauveria bassiana, B. caledonica, B. malawiensis, and B. pseudobassiana), to determine the most effective isolate for the control of DBM. Further assays then assessed whether selected combinations of these fungal isolates could increase the overall efficacy against DBM. Six Beauveria isolates (three B. bassiana and three B. pseudobassiana) achieved high DBM mortality at a low application rate with the first documented report of B. pseudobassiana able to kill 100% of DBM larvae. Further research determined that applications of low-virulent Beauveria isolates improved the control of DBM compared to mixtures containing high-virulent isolates. This novel approach increased the DBM pest mortality and shortened the time to kill.

Simple solution to preserve plant samples for microbiome analyses.

Culture-independent survey techniques are fundamental tools when assessing plant microbiomes. These methods rely on DNA that is carefully preserved after collecting samples to achieve meaningful results. Immediately freezing samples to -80°C after collection is considered one of the most robust methods for preserving samples before DNA extraction but is often impractical. Preservation solutions can solve this problem, but commercially available products are expensive, and there is limited data comparing their efficacy with other preservation methods. In this study, we compared the impact of three methods of sample preservation on plant microbiome surveys: (1) RNAlater, a proprietary preservative, (2) a home-made salt-saturated dimethyl sulphoxide preservation solution (DESS), and (3) freezing at -80°C. DESS-preserved samples, stored at room temperature for up to four weeks, did not show any significant differences to samples frozen at -80°C, while RNAlater inflated bacterial alpha diversity. Preservation treatments did not distinctively influence fungal alpha diversity. Our results demonstrate that DESS is a versatile and inexpensive preservative of DNA in plant material for diversity analyses of fungi and bacteria.

You are what you eat: fungal metabolites and host plant affect the susceptibility of diamondback moth to entomopathogenic fungi.

Background: Beauveria are entomopathogenic fungi of a broad range of arthropod pests. Many strains of Beauveria have been developed and marketed as biopesticides. Beauveria species are well-suited as the active ingredient within biopesticides because of their ease of mass production, ability to kill a wide range of pest species, consistency in different conditions, and safety with respect to human health. However, the efficacy of these biopesticides can be variable under field conditions. Two under-researched areas, which may limit the deployment of Beauveria-based biopesticides, are the type and amount of insecticidal compounds produced by these fungi and the influence of diet on the susceptibility of specific insect pests to these entomopathogens. Methods: To understand and remedy this weakness, we investigated the effect of insect diet and Beauveria-derived toxins on the susceptibility of diamondback moth larvae to Beauveria infection. Two New Zealand-derived fungal isolates, B. pseudobassiana I12 Damo and B. bassiana CTL20, previously identified with high virulence towards diamondback moth larvae, were selected for this study. Larvae of diamondback moth were fed on four different plant diets, based on different types of Brassicaceae, namely broccoli, cabbage, cauliflower, and radish, before their susceptibility to the two isolates of Beauveria was assessed. A second experiment assessed secondary metabolites produced from three genetically diverse isolates of Beauveria for their virulence towards diamondback moth larvae. Results: Diamondback moth larvae fed on broccoli were more susceptible to infection by B. pseudobassiana while larvae fed on radish were more susceptible to infection by B. bassiana. Furthermore, the supernatant from an isolate of B. pseudobassiana resulted in 55% and 65% mortality for half and full-strength culture filtrates, respectively, while the filtrates from two other Beauveria isolates, including a B. bassiana isolate, killed less than 50% of larvae. This study demonstrated different levels of susceptibility of the insects raised on different plant diets and the potential use of metabolites produced by Beauveria isolates in addition to their conidia.

Epichloë seed transmission efficiency is influenced by plant defense response mechanisms.

Asexual Epichloë are endophytic fungi that form mutualistic symbioses with cool-season grasses, conferring to their hosts protection against biotic and abiotic stresses. Symbioses are maintained between grass generations as hyphae are vertically transmitted from parent to progeny plants through seed. However, endophyte transmission to the seed is an imperfect process where not all seeds become infected. The mechanisms underpinning the varying efficiencies of seed transmission are poorly understood. Host gene expression in response to Epichloë sp. LpTG-3 strain AR37 was examined within inflorescence primordia and ovaries of high and low endophyte transmission genotypes within a single population of perennial ryegrass. A genome-wide association study was conducted to identify population-level single nucleotide polymorphisms (SNPs) and associated genes correlated with vertical transmission efficiency. For low transmitters of AR37, upregulation of perennial ryegrass receptor-like kinases and resistance genes, typically associated with phytopathogen detection, comprised the largest group of differentially expressed genes (DEGs) in both inflorescence primordia and ovaries. DEGs involved in signaling and plant defense responses, such as cell wall modification, secondary metabolism, and reactive oxygen activities were also abundant. Transmission-associated SNPs were associated with genes for which gene ontology analysis identified "response to fungus" as the most significantly enriched term. Moreover, endophyte biomass as measured by quantitative PCR of Epichloë non-ribosomal peptide synthetase genes, was significantly lower in reproductive tissues of low-transmission hosts compared to high-transmission hosts. Endophyte seed-transmission efficiency appears to be influenced primarily by plant defense responses which reduce endophyte colonization of host reproductive tissues.

Antagonism to Plant Pathogens by Epichloë Fungal Endophytes-A Review.

Epichloë is a genus of filamentous fungal endophytes that has co-evolved with cool-season grasses with which they form long-term, symbiotic associations. The most agriculturally important associations for pasture persistence for grazing livestock are those between asexual vertically transmitted Epichloë strains and the pasture species, perennial ryegrass, and tall fescue. The fungus confers additional traits to their host grasses including invertebrate pest deterrence and drought tolerance. Selected strains of these mutualistic endophytes have been developed into highly efficacious biocontrol products and are widely utilized within the Americas, Australia, and New Zealand for pasture persistence. Less publicized is the antagonism Epichloë endophytes display towards multiple species of saprophytic and pathogenic microbes. This opinion piece will review the current literature on antimicrobial properties exhibited by this genus of endophyte and discuss the reasons why this trait has historically remained a research curiosity rather than a trait of commercial significance.

Development of Plant-Fungal Endophyte Associations to Suppress Phoma Stem Canker in Brassica.

Endophytic microorganisms are found within the tissues of many plants species, with some conferring several benefits to the host plant including resistance to plant diseases. In this study, two putative endophytic fungi that were previously isolated from wild seeds of Brassica, identified as Beauveria bassiana and Pseudogymnoascus pannorum, were inoculated into cultivars of three Brassica species-Brassica napus, Br. rapa and Br. oleracea. Both fungal endophytes were reisolated from above- and below-ground tissues of inoculated plants at four different plant-growth stages, including cotyledon, one-leaf, two-leaf, and four-leaf stages. None of the plants colonised by these fungi exhibited any obvious disease symptoms, indicating the formation of novel mutualistic associations. These novel plant-endophyte associations formed between Brassica plants and Be. bassiana significantly inhibited phoma stem canker, a devastating disease of Brassica crops worldwide, caused by the fungal pathogen Leptosphaeria maculans. The novel association formed with P. pannorum significantly suppressed the amount of disease caused by L. maculans in one out of two experiments. Although biological control is not a new strategy, endophytic fungi with both antiinsect and antifungal activity are a highly conceivable, sustainable option to manage pests and diseases of economically important crops.

Symbiotic bacteria of plant-associated fungi: friends or foes?

Many bacteria form symbiotic associations with plant-associated fungi. The effects of these symbionts on host fitness usually depend on symbiont or host genotypes and environmental conditions. However, bacterial endosymbionts, that is those living within fungal cells, may positively regulate host performance as their survival is often heavily dependent on host fitness. Contrary to this, bacteria that establish ectosymbiotic associations with fungi, that is those located on the hyphal surface or in close vicinity to fungal mycelia, may not have an apparent net effect on fungal performance due to the low level of fitness dependency on their host. Our analysis supports the hypothesis that endosymbiotic bacteria of fungi are beneficial symbionts, and that effects of ectosymbiotic bacteria on fungal performance depends on the bacterial type involved in the interaction (e.g. helper versus pathogen of fungi). Ecological scenarios, where the presence of beneficial bacterial endosymbionts of fungi could be compromised, are also discussed.

Complete Genome Sequence of Paenibacillus sp. Strain E222, a Bacterial Symbiont of an Epichloë Fungal Endophyte of Ryegrass.

We report on the whole-genome sequence of Paenibacillus sp. strain E222, a bacterium isolated from a fresh culture of Epichloë festucae var. lolii, a mutualistic fungal endophyte of perennial ryegrass. The genome has a size of 7.8 Mb and a G+C content of 46% and encodes 6,796 putative protein-coding genes.

Methylobacterium, a major component of the culturable bacterial endophyte community of wild Brassica seed.

BACKGROUND: Plants are commonly colonized by a wide diversity of microbial species and the relationships created can range from mutualistic through to parasitic. Microorganisms that typically form symptomless associations with internal plant tissues are termed endophytes. Endophytes associate with most plant species found in natural and managed ecosystems. They are extremely important plant partners that provide improved stress tolerance to the host compared with plants that lack this symbiosis. Plant domestication has reduced endophyte diversity and therefore the wild relatives of many crop species remain untapped reservoirs of beneficial microbes. Brassica species display immense diversity and consequently provide the greatest assortment of products used by humans from a single plant genus important for agriculture, horticulture, bioremediation, medicine, soil conditioners, composting crops, and in the production of edible and industrial oils. Many endophytes are horizontally transmitted, but some can colonize the plant's reproductive tissues, and this gives these symbionts an efficient mechanism of propagation via plant seed (termed vertical transmission). METHODS: This study surveyed 83 wild and landrace Brassica accessions composed of 14 different species with a worldwide distribution for seed-originating bacterial endophytes. Seed was stringently disinfected, sown within sterile tissue culture pots within a sterile environment and incubated. After approximately 1-month, direct isolation techniques were used to recover bacterial endophytes from roots and shoots of symptomless plants. Bacteria were identified based on the PCR amplification of partial 16S rDNA gene sequences and annotated using the BLASTn program against the NCBI rRNA database. A diversity index was used as a quantitative measure to reflect how many different bacterial species there were in the seed-originating microbial community of the Brassica accessions sampled. RESULTS: Bacterial endophytes were recovered from the majority of the Brassica accessions screened. 16S rDNA gene sequencing identified 19 different bacterial species belonging to three phyla, namely Actinobacteria, Firmicutes and Proteobacteria with the most frequently isolated species being Methylobacterium fujisawaense, Stenotrophomonas rhizophila and Pseudomonas lactis. Methylobacterium was the dominant genus composing 56% of the culturable isolated bacterial community and was common in 77% of accessions possessing culturable bacterial endophytes. Two selected isolates of Methylobacterium significantly promoted plant growth when inoculated into a cultivar of oilseed rape and inhibited the growth of the pathogen Leptosphaeria maculans in dual culture. This is the first report that investigates the seed-originating endophytic microorganisms of wild Brassica species and highlights the Brassica microbiome as a resource for plant growth promoting bacteria and biological control agents.

A Tale of Two Grass Species: Temperature Affects the Symbiosis of a Mutualistic Epichloë Endophyte in Both Tall Fescue and Perennial Ryegrass.

Many cool-season grasses form permanent, mutualistic symbioses with asexual Epichloë endophytes. These fungal symbionts often perform a protective role within the association as many strains produce secondary metabolites that deter certain mammalian and invertebrate herbivores. Although initially a serious issue for agriculture, due to mammalian toxins that manifested in major animal health issues, selected strains that provide abiotic stress protection to plants with minimal ill effects to livestock are now commercialized and routinely used to enhance pasture performance in many farming systems. These fungal endophytes and their grass hosts have coevolved over millions of years, and it is now generally accepted that most taxonomic groupings of Epichloë are confined to forming compatible associations (i.e., symptomless associations) with related grass genera within a tribe. The most desired compounds associated with Epichloë festucae var. lolii, an endophyte species associated with perennial ryegrass, are peramine and epoxy-janthitrems. No other major secondary metabolites with invertebrate bioactivity have been identified within this association. However, other agriculturally beneficial compounds, such as lolines, have been discovered in related endophyte species that form associations with fescue grasses. A rationale therefore existed to develop novel grass-endophyte associations between loline-producing endophytes originally isolated from tall fescue with elite cultivars of perennial ryegrass to achieve a wider spectrum of insect bioactivity. A suitable loline-producing endophyte strain of Epichloë sp. FaTG-3 was selected and inoculated into perennial ryegrass. We hypothesed that endophyte transmission frequency, endophyte mycelial biomass and endophyte-derived alkaloid production would differ between the original tall fescue host and the artificial association. Consistent with our hypothesis, our data strongly suggest that plant species significantly affected the plant-endophyte association. This effect became more apparent for transmission frequency and endophyte biomass as the plants matured. Overall, the viable endophyte infection frequency was greater in the tall fescue host than in perennial ryegrass, at all sampling dates. Additionally, temperature was found to be a significant factor affecting endophyte transmission frequency, endophyte mycelial biomass and alkaloid production. Implications for the development of novel grass-endophyte associations are discussed.

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.