Leopoldo J. Iannone

Phylogenetic divergence, morphological and physiological differences distinguish a new Neotyphodium endophyte species in the grass Bromus auleticus from South America

The fungi of genus Neotyphodium are systemic, constitutive, symbionts of grasses of subfamily Pooideae. In the southern hemisphere most of these asexual endophytes are the result of the hybridization between two sexual species, Epichloë festucae and E. typhina, from the northern hemisphere. However the ancestral sexual species have not been detected in this region. Several grasses from Argentina are infected by Neotyphodium species. These endophytes are in general very similar macro-and micromorphologically and phylogenetically conform to species N. tembladerae. However the Neotyphodium spp. endophytes of some hosts, Bromus auleticus and Poa spicifomis var. spiciformis, have not been included in this species. In this work we studied the incidence and characterized the diversity of Neotyphodium species in populations of the native grass Bromus auleticus from Argentina. The incidence of endophytes was 100% in all populations investigated. Two groups of endophytes were differentiated by their morphologies, growth rates, conidial ontogenies and by relative resistance to the fungicide benomyl. Phylogenetic trees inferred from tefA and tubB intron sequences indicated that both N. tembladerae and the novel morphotype were hybrids of E. festucae and E. typhina, but the ancestral E. typhina genotype distinguished them. Isolates from plants that inhabit coastal dunes, xerophytic forests, savannahs and hills were similar morphologically and phylogenetically to N. tembladerae, whereas the endophytes from the humid pampa plains conformed to the novel group. We propose the endophyte of Bromus auleticus from humid pampas as a new species, Neotyphodium pampeanum.

Positive association between mycorrhiza and foliar endophytes in Poa bonariensis , a native grass

The interaction between mycorrhiza and leaf endophytes (Neotyphodium sp.) was studied in three Poa bonariensis populations, a native grass, differing significantly in endophyte infection. The association between endophytes and mycorrhizal fungi colonisation was assessed by analysing plant roots collected from the field. We found that roots from endophyte-infected populations showed a significantly higher frequency of colonisation by mycorrhizal fungi and that soil parameters were not related to endophyte infection or mycorrhiza colonization. In addition, we did not observe significant differences in the number of AM propagules in soils of the three populations sites. We also report the simultaneous development of Paris-type and Arum-type mycorrhiza morphology within the same root systems of P. bonariensis. The co-occurrence of both colonisation types in one and the same root system found in the three populations, which differed in Neotyphodium infection, suggests that foliar endophytes do not determine AM morphology. The percentage of root length colonised by different types of fungal structures (coils, arbuscules, longitudinal hyphae and vesicles) showed significant and positive differences in arbuscular frequency associated with endophyte infection, whereas the much smaller amounts of vesicles and hyphal coils did not differ significantly.

Evidence for leaf endophyte regulation of root symbionts: effect of Neotyphodium endophytes on the pre-infective state of mycorrhizal fungi

Neotyphodium endophytes and arbuscular mycorrhizal (AM) fungi are common constituents of natural grasslands. The plant–endophyte symbiosis can introduce changes in soil conditions that affect the density and activity of different functional groups of soil organisms. In the present work we performed in vitro assays to evaluate the effect of root and endophyte exudates on the pre-infective state of mycorrhizal fungi (Gigaspora margarita and G. rosea). Plant roots of Bromus setifolius from populations of Patagonia, and four strains of Neotyphodium were used to obtain the exudates. Root exudates of infected plants, at a high concentration, significantly increased AMF hyphal branches and length relative to exudates from naturally endophyte free plants. The effect of Neotyphodium endophyte exudates on AMF mycelial length varied depending on strain and the concentration used, suggesting a differential interaction between endophyte and AMF species. AMF hyphal branches were increased by Neotyphodium fungal exudates in both mycorrhizal species. A few previous studies have suggested that Neotyphodium endophytes can reduce mycorrhizal sporulation and colonization of host roots in commonly-cultivated agronomic hosts. In this study we report the opposite effect in B. setifolius. This study reports the direct and positive effect of root exudates from plants in symbiosis with Neotyphodium, on AMF pre-infective state. Further, identical effects were detected using exudates from Neotyphodium endophytes.

Diversity and distribution of Neotyphodium-infected grasses in Argentina

In the present work, we studied the presence of “epichloë/neotyphodium endophytes” in native grasses from Argentina. An extensive area of this country, representative of several different environments, was studied. Nine new host species of the genera Briza, Bromus and Poa were observed to be infected with asexual seed-borne endophytes. Epichloë stromata were not observed on any grass species. The incidence of infection in natural populations was highly variable among host species and among populations of the same host species. Morphological characterization revealed differences among the endophytes of different host species and among endophytes of different populations of the same host species. We also summarize the previous knowledge and present unpublished data on host diversity and distribution of these fungi in Argentina. Our results are discussed and compared with previous studies on endophytes in Argentina. This work supports the hypothesis that sexual species (Epichloë) are not present in the southern hemisphere, and suggests the existence of a high diversity of asexual endophytes in South America.

Species diversity of Epichloë symbiotic with two grasses from southern Argentinean Patagonia

In this work we performed morphological and molecular phylogenetic analyses (based on sequences of calmodulin M [calM], translation-elongation factor 1-α [tefA] and β-tubulin [tubB] genes) to characterize the diversity of Epichloë endophytes in Bromus setifolius and Phleum alpinum. The phylogenies obtained from the three genes were congruent and allowed differentiation of three lineages of endophytes that also presented morphological differences. One lineage corresponds to the previously described species Epichloë tembladerae, which is present in a wide range of native grasses from Argentina including B. setifolius and P. alpinum. Another genotype isolated only from B. setifolius is a non-hybrid endophyte, a rare condition for the South American Epichloë endophytes. Isolates of this genotype, described herein as a new variety, Epichloë typhina var. aonikenkana, presented waxy colonies at maturity and a low production of conidia. The third lineage, exclusively found in isolates from P. alpinum, is a hybrid between E. typhina and a common ancestor of E. amarillans and E. baconii. Isolates of this lineage produce abundant conidia that are variable in shape and size. Based on its unique phylogenetic position and morphology, we propose the new species, Epichloë cabralii for this lineage. The new combinations Epichloë tembladerae and E. pampeana also are proposed for the previously described Neotyphodium tembladerae and Neotyphodium pampeanum species.

The PAMPA datasets: a metagenomic survey of microbial communities in Argentinean pampean soils

BackgroundSoil is among the most diverse and complex environments in the world. Soil microorganisms play an essential role in biogeochemical cycles and affect plant growth and crop production. However, our knowledge of the relationship between species-assemblies and soil ecosystem processes is still very limited. The aim of this study was to generate a comprehensive metagenomic survey to evaluate the effect of high-input agricultural practices on soil microbial communities.ResultsWe collected soil samples from three different areas in the Argentinean Pampean region under three different types of land uses and two soil sources (bulk and rhizospheric). We extracted total DNA from all samples and also synthetized cDNA from rhizospheric samples. Using 454-FLX technology, we generated 112 16S ribosomal DNA and 14 16S ribosomal RNA amplicon libraries totaling 1.3 M reads and 36 shotgun metagenome libraries totaling 17.8 million reads (7.7 GB). Our preliminary results suggested that water availability could be the primary driver that defined microbial assemblages over land use and soil source. However, when water was not a limiting resource (annual precipitation >800 mm) land use was a primary driver.ConclusionThis was the first metagenomic study of soil conducted in Argentina and our datasets are among the few large soil datasets publicly available. The detailed analysis of these data will provide a step forward in our understanding of how soil microbiomes respond to high-input agricultural systems, and they will serve as a useful comparison with other soil metagenomic studies worldwide.

Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol

The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Effect of epichloid endophytes and soil fertilization on arbuscular mycorrhizal colonization of a wild grass

Background and aimsPlants often establish multiple simultaneous symbiotic associations with different micro-organisms; however, the way in which each symbiont affects the other symbionts and the effects of these multiple interactions on plant performance are not well understood. The aim of this study was to evaluate how two different asexual Epichloë species modulate the establishment of arbuscular mycorrhizal fungi (AMF) in a wild forage grass under different soil fertilization levels.MethodsWe performed a completely randomized 12-month-long field experiment to evaluate the effect of two B. auleticus-endophyte ecotypes and two soil fertilization levels on the colonization of AMF, in seedlings and adult plants. Plant biomass and reproductive tillers production were also measured.ResultsThe symbiosis, measured as the total extent of AM fungal colonization and frequency of arbuscules was significantly higher in Epichloë-infected plants and was not affected by fertilization either in seedlings or in adult plants. Plant biomass was increased by fertilization but no differences were observed due to the endophytic status. However, E+ plants produced more panicles than their E− counterparts.ConclusionsOur findings strongly support the hypothesis of positive association between Epichloë endophytes and AMF in wild grasses, making this model important for agronomic improvement.

Epichloë exudates promote in vitro and in vivo arbuscular mycorrhizal fungi development and plant growth

Background and aimsWe studied, through exudates employment, the effect of Epichloë (endophytic fungi), both independently and in association with Bromus auleticus (grass), on arbuscular mycorrhizal fungi (AMF) colonization, host and neighbouring plants biomass production and soil changes.MethodsThrough in vitro and greenhouse experiments, Epichloë endophytes effect on AMF development was evaluated. In vitro studies of exudates effect on Gigaspora rosea and Rhizophagus intraradices were performed using root or endophyte exudates. A 6-month greenhouse experiment was conducted to determine Bromus auleticus endophytic status effect and endophyte exudates role in biomass production, neighbouring plants mycorrhizal colonization and soil properties.ResultsEndophyte exudates and E+ plant root exudates promoted in vitro AMF development in the pre-infective stage of G. rosea and in carrot root culture mycelium of R. intraradices in a dose-response relationship, while control media and E- plants exudates had no effect. R. intraradices colonization and plant growth was clearly increased by endophytes and their exudates.ConclusionsThis is the first work evidencing the direct effect of Epichloë endophytes and infected plants root exudates on AMF extramatrical development. While higher levels of AMF colonization were observed in E+ plants, no clear effect was detected in neighbouring plants colonization, plant biomass or soil properties.