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.

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.

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.

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.

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.

Prevalence and diversity of viruses in the entomopathogenic fungus Beauveria bassiana.

Viruses have been discovered in numerous fungal species, but unlike most known animal or plant viruses, they are rarely associated with deleterious effects on their hosts. The knowledge about viruses among entomopathogenic fungi is very limited, although their existence is suspected because of the presence of virus-like double-stranded RNA (dsRNA) in isolates of several species. Beauveria bassiana is one of the most-studied species of entomopathogenic fungi; it has a cosmopolitan distribution and is used as a biological control agent against invertebrates in agriculture. We analyzed a collection of 73 isolates obtained at different locations and from different habitats in Spain and Portugal, searching for dsRNA elements indicative of viral infections. The results revealed that the prevalence of viral infections is high; 54.8% of the isolates contained dsRNA elements with viral characteristics. The dsRNA electropherotypes of infected isolates indicated that virus diversity was high in the collection analyzed and that mixed virus infections occurred in fungal isolates. However, a hybridization experiment indicated that dsRNA bands that are similar in size do not always have similar sequences. Particular virus species or dsRNA profiles were not associated with locations or types of habitats, probably because of the ubiquity and efficient dispersion of this fungus as an airborne species. The sequence of one of the most common dsRNA elements corresponded to the 5.2-kbp genome of a previously undescribed member of the Totiviridae family, termed B. bassiana RNA virus 1 (BbRV1).

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.

A spectroscopy approach to the study of virus infection in the endophytic fungus Epichloë festucae.

BACKGROUND: In this work we propose a rapid method based on visible and near-infrared (Vis-NIR) spectroscopy to determine the occurrence of double-stranded RNA (dsRNA) viruses in Epichloë festucae strains isolated from Festuca rubra plants. In addition, we examined the incidence of infections by E. festucae in populations of F. rubra collected in natural grasslands of Western Spain. METHODS: Vis-NIR spectra (400-2498 nm) from 124 virus-infected and virus-free E. festucae isolates were recorded directly from ground and freeze-dried mycelium. To estimate how well the spectra for uninfected and infected fungal samples could be differentiated, we used partial least-squares discriminant analysis (PLS1-DA) and several data pre-treatments to develop calibration models. RESULTS: Applying the best regression model, obtained with two sampling years and using standard normal variate (SNV) combined with first derivative transformation to a new validating data set (42 samples), we obtained a correct classification for 75% of the uninfected isolates and up to 86% of the infected isolates. CONCLUSIONS: The results obtained suggest that Vis-NIR spectroscopy is a promising technology for detection of viral infections in fungal samples when an alternative faster approach is desirable. It provides a tool adequately exact and more time- and cost-saving than the conventional reference analysis.

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.

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.

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.

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.

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.

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.

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.

Mycoviruses are common among different species of endophytic fungi of grasses.

A survey of mycoviruses was made in a collection of 103 isolates belonging to 53 different species of endophytic fungi of grasses. Double-stranded RNA (dsRNA) elements were detected in isolates of 12 of the species analyzed. The banding characteristics and sizes of some of the dsRNA elements suggest that they might belong to previously described mycovirus families. The observed incidence (22.6%) indicates that the presence of mycoviruses could be common among species of this group of ubiquitous fungi.

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.