Symbiotic fungi from a wild grass (Celtica gigantea) increase the growth, grain yield and quality of tritordeum under field conditions.

Plants function in symbiosis with numerous microorganisms, which might contribute to their adaptation and performance. In this study, we tested whether fungal strains in symbiotic interaction with roots of Celtica gigantea, a wild grass adapted to nutrient-poor soils in semiarid habitats, could improve the field performance of the agricultural cereal tritordeum (Triticum durum × Hordeum chilense). Seedlings of tritordeum were inoculated with 12 different fungal strains isolated from roots of Celtica gigantea that were first proved to promote the growth of tritordeum plants under greenhouse conditions. The inoculated seedlings were transplanted to field plots at two locations belonging to different climatic zones in terms of mean temperatures and precipitation in the Iberian Peninsula. Only one strain, Diaporthe iberica T6, had a significant effect on plant height, number of tillers and grain yield in one location. This result showed a substantial divergence between the results of greenhouse and field tests. In terms of grain nutritional quality, several parameters were differentially affected at both locations: Diaporthe T6, Pleosporales T7, Zygomycota T29 and Zygomycota T80 increased the content of total carotenoids, mainly lutein, in the colder location; whereas gluten proteins increased with several treatments in the warmer location. In conclusion, early inoculation of tritordeum plants with fungal symbionts had substantial beneficial effects on subsequent plant growth and development in the field. Regarding grain nutritional quality, the effect of inoculation was affected by the agroclimatic differences between both field locations.

Diaporthe species associated with the maritime grass Festuca rubra subsp. pruinosa.

Festuca rubra subsp. pruinosa is a perennial grass growing in sea cliffs where plants are highly exposed to salinity and marine winds, and often grow in rock fissures where soil is absent. Diaporthe species are one of the most abundant components of the root microbiome of this grass and several Diaporthe isolates have been found to produce beneficial effects in their host and other plant species of agronomic importance. In this study, 22 strains of Diaporthe isolated as endophytes from roots of Festuca rubra subsp. pruinosa were characterized by molecular, morphological, and biochemical analyses. Sequences of the nuclear ribosomal internal transcribed spacers (ITS), translation elongation factor 1-α (TEF1), beta-tubulin (TUB), histone-3 (HIS), and calmodulin (CAL) genes were analyzed to identify the isolates. A multi-locus phylogenetic analysis of the combined five gene regions led to the identification of two new species named Diaporthe atlantica and Diaporthe iberica. Diaporthe atlantica is the most abundant Diaporthe species in its host plant, and Diaporthe iberica was also isolated from Celtica gigantea, another grass species growing in semiarid inland habitats. An in vitro biochemical characterization showed that all cultures of D. atlantica produced indole-3-acetic acid and ammonium, and the strains of D. iberica produced indole 3-acetic acid, ammonium, siderophores, and cellulase. Diaporthe atlantica is closely related to D. sclerotioides, a pathogen of cucurbits, and caused a growth reduction when inoculated in cucumber, melon, and watermelon.

Diaporthe atlantica enhances tomato drought tolerance by improving photosynthesis, nutrient uptake and enzymatic antioxidant response.

Functional symbiosis with fungal endophytes can help plants adapt to environmental stress. Diaporthe atlantica is one of the most abundant fungal taxa associated with roots of Festuca rubra subsp. pruinosa, a grass growing in sea cliffs. This study aimed to investigate the ability of a strain of this fungus to ameliorate the impact of drought stress on tomato plants. In a greenhouse experiment, tomato plants were inoculated with Diaporthe atlantica strain EB4 and exposed to two alternative water regimes: well-watered and drought stress. Several physiological and biochemical plant parameters were evaluated. Inoculation with Diaporthe promoted plant growth in both water treatments. A significant interactive effect of Diaporthe-inoculation and water-regime showed that symbiotic plants had higher photosynthetic capacity, water-use efficiency, nutrient uptake (N, P, K, Fe and Zn), and proline content under drought stress, but not under well-watered conditions. In addition, Diaporthe improved the enzymatic antioxidant response of plants under drought, through an induced mechanism, in which catalase activity was modulated and conferred protection against reactive oxygen species generation during stress. The results support that Diaporthe atlantica plays a positive role in the modulation of tomato plant responses to drought stress by combining various processes such as improving photosynthetic capacity, nutrient uptake, enzymatic antioxidant response and osmo-protectant accumulation. Thus, drought stress in tomato can be enhanced with symbiotic fungi.

A Diaporthe Fungal Endophyte From a Wild Grass Improves Growth and Salinity Tolerance of Tritordeum and Perennial Ryegrass.

Some microbiome components can provide functions that extend the capabilities of plants, increasing the environmental adaptability and performance of holobionts. Festuca rubra subsp. pruinosa is a perennial grass adapted to rocky sea cliffs, where soil and nutrients are very limited, and exposure to salinity is continuous. This study aimed to investigate if a Diaporthe fungal endophyte belonging to the core microbiome of Festuca rubra roots could improve the performance of two agricultural grasses. In a greenhouse experiment, plants of tritordeum (Triticum durum x Hordeum chilense) and perennial ryegrass (Lolium perenne) were inoculated with Diaporthe strain EB4 and subjected to two salinity conditions (0 and 200 mM NaCl). Biomass production, mineral elements, proline, hormone profiles, antioxidant capacity, and total phenolic compounds were examined in plants, and fungal functions potentially related to the promotion of plant growth were determined. The inoculation with Diaporthe promoted plant growth of both grasses, increasing leaf biomass (84% in tritordeum and 29% in perennial ryegrass), root biomass, nutrient content (N, Ca, Mg, and Fe), and the production of indole 3-acetic acid, regardless of the salinity treatment. Improved growth and nutrient uptake might occur because Diaporthe produces several extracellular enzymes capable of recycling organic nutrient pools. In addition, the fungus produced indole 3-acetic acid in vitro and modulated the production of this phytohormone in the plant. Under salinity, the activity of Diaporthe ameliorated the stress, increasing proline, nutrient uptake in roots, gibberellins, and indole 3-acetic acid, which in turn results into improved growth. Thus, this fungus can transfer to alternative hosts some advantages useful at its original habitat.

Genetic Diversity of the Symbiotic Fungus Epichloë festucae in Naturally Occurring Host Grass Populations.

Epichloë festucae is a common symbiont of the perennial and widely distributed cool season grass, Festuca rubra. The symbiosis is highly integrated involving systemic growth of the fungus throughout above-ground host parts and vertical transmission from plant to its offspring via host seeds. However, the nature of symbiosis is labile ranging from antagonistic to mutualistic depending on prevailing selection pressures. Both the loss of fungus in the maternal host lineage and horizontal transmission through sexual spores within the host population may partly explain the detected variation in symbiosis in wild grass populations. Epichloë species are commonly considered as pathogens when they produce sexual spores and partly castrate their host plant. This is the pathogenic end of the continuum from antagonistic to mutualistic interactions. Here we examined the population genetic structure of E. festucae to reveal the gene flow, importance of reproduction modes, and alkaloid potential of the symbiotic fungus in Europe. Epichloë-species are highly dependent on the host in survival and reproduction whilst benefits to the host are largely linked to defensive mutualism attributable to fungal-origin bioactive alkaloids that negatively affect vertebrate and/or invertebrate herbivores. We detected decreased genetic diversity in previously glaciated areas compared to non-glaciated regions during the last glacial maximum period and found three major genetic clusters in E. festucae populations: southern, northeastern and northwestern Europe. Sexual reproduction may have a higher role than expected in Spanish E. festucae populations due to the predominance of unique genotypes and presence of both mating types in the region. In contrast, asexual reproduction via host seeds predominates in the Faroe Island and Finland in northern Europe due to the presence of biased mating-type ratios and large dominant genotypes in the E. festucae populations within the region. A substantially larger variation of alkaloid genotypes was observed in the fungal populations than expected, although the variability of the alkaloid genotypes within populations is considerably lower in northern than Spanish populations in southern Europe. E. festucae populations consist of different combinations of alkaloid classes from the gene clusters of ergot alkaloid and indole-terpenes, and from pyrrolopyrazine alkaloid gene. We suggest that the postglacial distribution history of the host grass, prevailing reproduction strategies of E. festucae, and local selection pressures likely explain a large part of the genetic variation observed in fungal populations among geographic regions. The identified alkaloid genotypes can be used by turfgrass breeders to improve resistance against herbivores in red fescue varieties and to develop new sustainable cultivars in Europe.

The Role of Fungal Microbiome Components on the Adaptation to Salinity of Festuca rubra subsp. pruinosa.

Festuca rubra subsp. pruinosa is a perennial grass that inhabits sea cliffs, a habitat where salinity and low nutrient availability occur. These plants have a rich fungal microbiome, and particularly common are their associations with Epichloë festucae in aboveground tissues and with Fusarium oxysporum and Periconia macrospinosa in roots. In this study, we hypothesized that these fungi could affect the performance of F. rubra plants under salinity, being important complements for plant habitat adaptation. Two lines of F. rubra, each one consisting of Epichloë-infected and Epichloë-free clones, were inoculated with the root endophytes (F. oxysporum and P. macrospinosa) and subjected to a salinity treatment. Under salinity, plants symbiotic with Epichloë had lower Na+ content than non-symbiotic plants, but this effect was not translated into plant growth. P. macrospinosa promoted leaf and root growth in the presence and absence of salinity, and F. oxysporum promoted leaf and root growth in the presence and absence of salinity, plus a decrease in leaf Na+ content under salinity. The growth responses could be due to functions related to improved nutrient acquisition, while the reduction of Na+ content might be associated with salinity tolerance and plant survival in the long term. Each of these three components of the F. rubra core mycobiome contributed with different functions, which are beneficial and complementary for plant adaptation to its habitat in sea cliffs. Although our results do not support an obvious role of Epichloë itself in FRP salt tolerance, there is evidence that Epichloë can interact with root endophytes, affecting host plant performance.

Disruption of Traditional Grazing and Fire Regimes Shape the Fungal Endophyte Assemblages of the Tall-Grass Brachypodium rupestre.

The plant microbiome is likely to play a key role in the resilience of communities to the global climate change. This research analyses the culturable fungal mycobiota of Brachypodium rupestre across a sharp gradient of disturbance caused by an intense, anthropogenic fire regime. This factor has dramatic consequences for the community composition and diversity of high-altitude grasslands in the Pyrenees. Plants were sampled at six sites, and the fungal assemblages of shoots, rhizomes, and roots were characterized by culture-dependent techniques. Compared to other co-occurring grasses, B. rupestre hosted a poorer mycobiome which consisted of many rare species and a few core species that differed between aerial and belowground tissues. Recurrent burnings did not affect the diversity of the endophyte assemblages, but the percentages of infection of two core species -Omnidemptus graminis and Lachnum sp. -increased significantly. The patterns observed might be explained by (1) the capacity to survive in belowground tissues during winter and rapidly spread to the shoots when the grass starts its spring growth (O. graminis), and (2) the location in belowground tissues and its resistance to stress (Lachnum sp.). Future work should address whether the enhanced taxa have a role in the expansive success of B. rupestre in these anthropized environments.

Untapping the potential of plant mycobiomes for applications in agriculture.

Plant-fungal interactions are widespread in nature, and their multiple benefits for plant growth and health have been amply demonstrated. Endophytic and epiphytic fungi can significantly increase plant resilience, improving plant nutrition, stress tolerance and defence. Although some of these interactions have been known for decades, the relevance of the plant mycobiome within the plant microbiome has been largely underestimated. Our limited knowledge of fungal biology and their interactions with plants in the broader phytobiome context has hampered the development of optimal biotechnological applications in agrosystems and natural ecosystems. Exciting recent technical and knowledge advances in the context of molecular and systems biology open a plethora of opportunities for developing this field of research.

Screening fungal endophytes from a wild grass for growth promotion in tritordeum, an agricultural cereal.

Celtica gigantea(= Stipa gigantea) is a large perennial grass which grows in nutrient-poor sandy soils in semiarid zones of the western Iberian Peninsula. The purpose of this work was to find out if culturable fungal symbionts isolated from roots of this wild grass could have growth promoting activity in tritordeum, a hybrid cereal for human consumption. A survey of fungi from the root endosphere of C. gigantea produced an isolate collection consisting of 60 different taxa, mostly ascomycetes. Fungal strains were inoculated into tritordeum plants in order to evaluate their effect in leaf and root biomass, nutrient content, and total antioxidant capacity. Two consecutive screening processes were made to test endophyte effects in plants. In the first screening, 66 strains were inoculated into seedlings by dipping roots in a liquid suspension of inoculum. In the second screening, 13 strains selected from the first screening were inoculated by sowing seeds in a substrate containing inoculum. The inoculation method used in the second screening involved less labor and plant manipulation and improved the quantity and quality of the inoculum, making it more appropriate for big scale experimental inoculation procedures. Several fungal strains promoted leaf or root growth. In particular, a strain belonging to the genus Diaporthe caused an increase in leaf and root biomass in both screening processes, suggesting that this endophyte might have a good potential for field application in tritordeum.

Epichloë Endophyte-Promoted Seed Pathogen Increases Host Grass Resistance Against Insect Herbivory.

Plants host taxonomically and functionally complex communities of microbes. However, ecological studies on plant-microbe interactions rarely address the role of multiple co-occurring plant-associated microbes. Here, we contend that plant-associated microbes interact with each other and can have joint consequences for higher trophic levels. In this study we recorded the occurrence of the plant seed pathogenic fungus Claviceps purpurea and aphids (Sitobion sp.) on an established field experiment with red fescue (Festuca rubra) plants symbiotic to a seed transmitted endophytic fungus Epichloë festucae (E+) or non-symbiotic (E-). Both fungi are known to produce animal-toxic alkaloids. The study was conducted in a semi-natural setting, where E+ and E- plants from different origins (Spain and Northern Finland) were planted in a randomized design in a fenced common garden at Kevo Subarctic Research Station in Northern Finland. The results reveal that 45% of E+ plants were infected with Claviceps compared to 31% of E- plants. Uninfected plants had 4.5 times more aphids than Claviceps infected plants. By contrast, aphid infestation was unaffected by Epichloë symbiosis. Claviceps alkaloid concentrations correlated with a decrease in aphid numbers, which indicates their insect deterring features. These results show that plant mutualistic fungi can increase the infection probability of a pathogenic fungus, which then becomes beneficial to the plant by controlling herbivorous insects. Our study highlights the complexity and context dependency of species-species and multi-trophic interactions, thus challenging the labeling of species as plant mutualists or pathogens.

Brassica oleracea var. acephala (kale) improvement by biological activity of root endophytic fungi.

Brassica oleracea var. acephala (kale) is a cruciferous vegetable widely cultivated for its leaves and flower buds in Atlantic Europe and the Mediterranean area, being a food of great interest as a "superfood" today. Little has been studied about the diversity of endophytic fungi in the Brassica genus, and there are no studies regarding kale. In this study, we made a survey of the diversity of endophytic fungi present in the roots of six different Galician kale local populations. In addition, we investigated whether the presence of endophytes in the roots was beneficial to the plants in terms of growth, cold tolerance, or resistance to bacteria and insects. The fungal isolates obtained belonged to 33 different taxa. Among those, a Fusarium sp. and Pleosporales sp. A between Setophoma and Edenia (called as Setophoma/Edenia) were present in many plants of all five local populations, being possible components of a core kale microbiome. For the first time, several interactions between endophytic fungus and Brassica plants are described and is proved how different interactions are beneficial for the plant. Fusarium sp. and Pleosporales sp. B close to Pyrenophora (called as Pyrenophora) promoted plant growth and increased cold tolerance. On the other hand, isolates of Trichoderma sp., Pleosporales sp. C close to Phialocephala (called as Phialocephala), Fusarium sp., Curvularia sp., Setophoma/Edenia and Acrocalymma sp. were able to activate plant systemic resistance against the bacterial pathogen Xanthomonas campestris. We also observed that Fusarium sp., Curvularia sp. and Setophoma/Edenia confered resistance against Mamestra brassicae larvae.

Cultivation and growth dynamics of endophytic fungi in a solid culture medium based on sugar beet pulp.

BACKGROUND: The production of mycelium from endophytic fungi is of interest for applications ranging from inoculants and biofertilizers for crop production to fermentations for enzyme and metabolite production. The purpose of this study was to test the capacity of a solid growth medium based on beet pulp for growing different strains of endophytes. RESULTS: The ergosterol content of inoculated medium was measured to estimate fungal growth. Several parameters related to the preparation of the growth medium, such as water content, calcium salts and incubation time, were evaluated. The greatest fungal biomass production was observed in a medium prepared with a 1:2 (beet pulp:water) ratio, containing calcium sulfate and carbonate. Strains belonging to different fungal species grew well in the growth medium finally selected, producing yields ranging from 50 to 500 g mycelium per kilogram of dry culture, after 22-27 days. Cultures containing up to 400 g beet pulp grew successfully, and could be scaled up. CONCLUSION: A solid culture medium based on beet pulp supported the growth of diverse taxa of fungal endophytes. Both the water and calcium salt content of the growth medium affected the efficiency of mycelium production. Considering these factors, beet pulp medium was an excellent endophyte cultivation medium because of the high yield of fungal biomass observed, together with its ease of handling and scaling-up production. © 2019 Society of Chemical Industry.

Local adaptation in natural European host grass populations with asymmetric symbiosis.

Recent work on microbiomes is revealing the wealth and importance of plant-microbe interactions. Microbial symbionts are proposed to have profound effects on fitness of their host plants and vice versa, especially when their fitness is tightly linked. Here we studied local adaptation of host plants and possible fitness contribution of such symbiosis in the context of abiotic environmental factors. We conducted a four-way multi-year reciprocal transplant experiment with natural populations of the perennial grass Festuca rubra s.l. from northern and southern Finland, Faroe Islands and Spain. We included F. rubra with and without transmitted symbiotic fungus Epichloë that is vertically transmitted via host seed. We found local adaptation across the European range, as evidenced by higher host fitness of the local geographic origin compared with nonlocals at three of the four studied sites, suggesting that selection pressures are driving evolution in different directions. Abiotic factors did not result in strong fitness effects related to Epichloë symbiosis, indicating that other factors such as herbivory are more likely to contribute to fitness differences between plants naturally occurring with or without Epichloë. Nevertheless, in the case of asymmetric symbiosis that is obligatory for the symbiont, abiotic conditions that affect performance of the host, may also cause selective pressure for the symbiont.

Conditioned media and organic elicitors underpin the production of potent antiplasmodial metabolites by endophytic fungi from Cameroonian medicinal plants.

Plasmodial resistance to artemisinin-based combination therapies emphasizes the need for new drug development to control malaria. This paper describes the antiplasmodial activity of metabolites produced by endophytic fungi of three Cameroonian plants. Ethyl acetate extracts of fungi cultivated on three different media were tested against Plasmodium falciparum chloroquine-sensitive (Pf3D7) and chloroquine-resistant (PfINDO) strains using the SYBR green florescence assay. Selected endophytes were further grown in potato dextrose broth supplemented with small organic elicitors and their extracts tested for activity. The effect of elicitors on de novo metabolite synthesis was assessed by reverse-phase HPLC. Activity screening of 81 extracts indicated that Aspergillus niger 58 (IC50 2.25-6.69 µg/mL, Pf3D7), Fusarium sp. N240 (IC50 1.62-4.38 µg/mL, Pf3D7), Phomopsis sp. N114 (IC50 0.34-7.26 µg/mL, Pf3D7), and Xylaria sp. N120 (IC50 2.69-6.77 µg/mL, Pf3D7) produced potent extracts when grown in all three media. Further culture of these endophytes in potato dextrose broth supplemented with each of the eight small organic elicitors and subsequent extracts screening indicated the extract of Phomopsis sp. N114 grown with 1% 1-butanol to be highly selective and extremely potent (IC50 0.20-0.33 µg/mL; SI > 666). RPHPLC profiles of extracts of Phomopsis sp. N114 grown with or without 1-butanol showed some peaks of enhanced intensities in the former without any qualitative change in the chromatograms. This study showed the ability of selected endophytes to produce potent and selective antiplasmodial metabolites in varied culture conditions. It also showed how the production of desired metabolites can be enhanced by use of small molecular weight elicitors.

Near-infrared spectroscopy allows detection and species identification of Epichloë endophytes in Lolium perenne.

BACKGROUND: Perennial ryegrass (Lolium perenne) is systemically infected by seed-transmitted fungal endophytes (Epichloë sp.). The presence of Epichloë endophytes alters the nutritive quality of its hosts by modifying several plant traits. The aim of this research was to develop a fast method based on near-infrared reflectance spectroscopy (NIRS) to discriminate between perennial ryegrass plants infected (E+) or not infected (E-) with two endophyte species, Epichloë festucae var. lolii, and Epichloë typhina, using a heterogonous set of perennial ryegrass samples collected from wild grasslands and cultivars. Epichloë festucae var. lolii cultures show two morphotypes, M1 and M3, and Epichloë typhina cultures have a different M2 morphotype. RESULTS: Near-infrared reflectance spectra from E+ and E- ryegrass plants were recorded. Applying the best NIRS model for the detection of Epichloë, 93.3% of E+ plants were classified correctly. The NIRS morphotype classification was correct for 92.9% of M1 morphotype and 100% of M2 morphotypes. The NIRS classification of M3 morphotypes was not as accurate, but it was in accordance with the fungal species classification, identifying some M3 as M1 morphotypes. CONCLUSION: Near-infrared reflectance spectroscopy can detect the presence of Epichloë fungal endophytes directly in samples of perennial ryegrass, and it is adequate for discriminating among fungal species. © 2018 Society of Chemical Industry.

Production of fumonisins by endophytic strains of Tolypocladium cylindrosporum and its relation to fungal virus infection.

Fumonisins were first discovered in Fusarium verticillioides, a fungus associated to disease and asymptomatic infections in maize. Afterwards, other fungal taxa have been found to produce fumonisins. The entomopathogenic ascomycete Tolypocladium cylindrosporum has been isolated from soil and also as an endophyte from leaves of grasses. The objectives of this work were to determine the in vitro production of fumonisin B (FB) mycotoxins and the immunosuppressive compound cyclosporine A (CyA) in several strains of T. cylindrosporum, and to examine the effect of fungal virus infection and temperature in FB production. FB1 was detected in 30% of the strains, ranging from 0.16 to 5.52 µg cm-2 in solid media, and FB2 was detected in 78% of the strains, ranging from 0.764 to 40.92 µg cm-2. CyA was not detected in any strain. The mean FB2 concentration of the endophytic strain Tc37W was three times greater (p < 0.05) than that of any other strain. Up to 34% more of FB2 was detected in strains infected by the virus TcV3 than in the corresponding virus-free versions. The effect of temperature on FB2 content was interactively significantly dependent on fungal strain and growth medium; in the YES medium, the FB2 of virus-infected strains Tc37-1V and Tc37W increased by 67 and 16%, respectively, at 26 °C as compared to 20 °C. The FB concentration in some fungal strains was similar to that in fungi associated to food and feed intoxications.

A Survey of Culturable Fungal Endophytes From Festuca rubra subsp. pruinosa, a Grass From Marine Cliffs, Reveals a Core Microbiome.

Festuca rubra subsp. pruinosa is a perennial grass that inhabits sea cliffs of the Atlantic coasts of Europe. In this unhospitable environment plants grow in rock crevices and are exposed to abiotic stress factors such as low nutrient availability, wind, and salinity. Festuca rubra subsp. pruinosa is a host of the fungal endophyte Epichloë festucae, which colonizes aerial organs, but its root mycobiota is unknown. The culturable endophytic mycobiota of FRP roots was surveyed in a set of 105 plants sampled at five populations in marine cliffs from the northern coast of Spain. In total, 135 different fungal taxa were identified, 17 of them occurred in more than 10% of plants and in two or more populations. Seven taxa belonging to Fusarium, Diaporthe, Helotiales, Drechslera, Slopeiomyces, and Penicillium appeared to be constituents of the core microbiome of Festuca rubra subsp. pruinosa roots because they occurred in more than 20% of the plants analyzed, and at three or more populations. Most fungal strains analyzed (71.8%) were halotolerant. The presence of Epichloë festucae in aboveground tissue was detected in 65.7% of the plants, but its presence did not seem to significantly affect the structure of the core or other root microbiota, when compared to that of plants free of this endophyte. When plants of the grass Lolium perenne were inoculated with fungal strains obtained from Festuca rubra subsp. pruinosa roots, a Diaporthe strain significantly promoted leaf biomass production under normal and saline (200 mM NaCl) watering regimes. These results suggest that the core mycobiome of Festuca rubra subsp. pruinosa could have a role in host plant adaptation, and might be useful for the improvement of agricultural grasses.

Qualitative and quantitative analysis of endophyte alkaloids in perennial ryegrass using near-infrared spectroscopy.

BACKGROUND: Near-infrared reflectance spectroscopy (NIRS) has been widely used in forage quality control because it is faster, cleaner and less expensive than conventional chemical procedures. In Lolium perenne (perennial ryegrass), one of the most important forage grasses, the infection by asymptomatic Epichloë fungal endophytes alters the plant nutritional quality due to the production of alkaloids. In this research, we developed a rapid method based on NIRS to detect and quantify endophyte alkaloids (peramine, lolitrem B and ergovaline) using a heterogeneous set of L. perenne plants obtained from wild grasslands and cultivars. RESULTS: NIR spectra from dried grass samples were recorded and classified according to the absence or presence of alkaloids, based on reference methods. The best discriminant equations for detection of alkaloids classified correctly 94.4%, 87.5% and 92.9% of plants containing peramine, lolitrem B and ergovaline, respectively. The quantitative NIR equations obtained by modified partial least squares (MPLS) algorithm had coefficients of correlation of 0.93, 0.41, and 0.76 for peramine, lolitrem B and ergovaline respectively. CONCLUSION: NIRS is a suitable tool for qualitative analysis of endophyte alkaloids in grasses and for the accurate quantification of peramine and ergovaline. © 2017 Society of Chemical Industry.

Solutions to decrease a systematic error related to AAPH addition in the fluorescence-based ORAC assay.

Oxygen radical absorbance capacity (ORAC) assay in 96-well multi-detection plate readers is a rapid method to determine total antioxidant capacity (TAC) in biological samples. A disadvantage of this method is that the antioxidant inhibition reaction does not start in all of the 96 wells at the same time due to technical limitations when dispensing the free radical-generating azo initiator 2,2'-azobis (2-methyl-propanimidamide) dihydrochloride (AAPH). The time delay between wells yields a systematic error that causes statistically significant differences in TAC determination of antioxidant solutions depending on their plate position. We propose two alternative solutions to avoid this AAPH-dependent error in ORAC assays.

Fungal endophyte (Epichloë festucae) alters the nutrient content of Festuca rubra regardless of water availability.

Festuca rubra plants maintain associations with the vertically transmitted fungal endophyte Epichloë festucae. A high prevalence of infected host plants in semiarid grasslands suggests that this association could be mutualistic. We investigated if the Epichloë-endophyte affects the growth and nutrient content of F. rubra plants subjected to drought. Endophyte-infected (E+) and non-infected (E-) plants of two half-sib lines (PEN and RAB) were subjected to three water availability treatments. Shoot and root biomass, nutrient content, proline, phenolic compounds and fungal alkaloids were measured after the treatments. The effect of the endophyte on shoot and root biomass and dead leaves depended on the plant line. In the PEN line, E+ plants had a greater S:R ratio than E-, but the opposite occurred in RAB. In both plant lines and all water treatments, endophyte-infected plants had greater concentrations of N, P and Zn in shoots and Ca, Mg and Zn in roots than E- plants. On average, E+ plants contained in their shoots more P (62%), Zn (58%) and N (19%) than E- plants. While the proline in shoots increased in response to water stress, the endophyte did not affect this response. A multivariate analysis showed that endophyte status and plant line impose stronger differences in the performance of the plants than the water stress treatments. Furthermore, differences between PEN and RAB lines seemed to be greater in E- than in E+ plants, suggesting that E+ plants of both lines are more similar than those of their non-infected version. This is probably due to the endophyte producing a similar effect in both plant lines, such as the increase in N, P and Zn in shoots. The remarkable effect of the endophyte in the nutrient balance of the plants could help to explain the high prevalence of infected plants in natural grasslands.