Navigating the labyrinth: a guide to sequence-based, community ecology of arbuscular mycorrhizal fungi

Data generated from next generation sequencing (NGS) will soon comprise the majority of information about arbuscular mycorrhizal fungal (AMF) communities. Although these approaches give deeper insight, analysing NGS data involves decisions that can significantly affect results and conclusions. This is particularly true for AMF community studies, because much remains to be known about their basic biology and genetics. During a workshop in 2013, representatives from seven research groups using NGS for AMF community ecology gathered to discuss common challenges and directions for future research. Our goal was to improve the quality and accessibility of NGS data for the AMF research community. Discussions spanned sampling design, sample preservation, sequencing, bioinformatics and data archiving. With concrete examples we demonstrated how different approaches can significantly alter analysis outcomes. Failure to consider the consequences of these decisions may compound bias introduced at each step along the workflow. The products of these discussions have been summarized in this paper in order to serve as a guide for any researcher undertaking NGS sequencing of AMF communities.

PHOSPHORUS DOSES DETERMINE THE PREVALENCE OF NATIVE ARBUSCULAR MYCORRHIZAL FUNGI IN Araucaria angustifolia

A greenhouse experiment was installed with bait cultures to extract the AMF species present in a rhizosphere soil sample of a native Araucaria angustifolia forest in Campos do Jordao, Brazil. The experimental design was completely randomized, with four increasing phosphorus doses (0, 20, 50, and 150 mg kg(-1), as triple superphosphate), with five replicates, the bait plant was araucaria, and all pots were inoculated with 100 g of rhizospheric soil collected in an araucaria forest. After twelve months the spores were extracted, counted and identified, and the percent root colonization was also determined. When taking all four P doses into account, eleven AMF species could be identified: Acaulospora bireticulata, Acaulospora morrowiae, Acaulospora sp., Entrophospora colombiana, Gigaspora margarita, Glomus diaphanum, Glomus etunicatum, Glomus macrocarpum, Scutellospora calospora, Scutellospora gilmorei, and Scutellospora pellucida. There was no effect of the P dose on the total amount of spores neither on the percent root colonization. However, the correspondence analysis showed that the different AMF species were selectively associated mostly to either one or another P dose.

Early responses of wild plant seedlings to arbuscular mycorrhizal fungi and pathogens

Abstract Many plants form associations with arbuscular mycorrhizal fungi (AMF) because they profit from improved phosphorus nutrition and from protection against pathogens. Whereas mycorrhiza-induced pathogen protection is well understood in agricultural plant species, it is rarely studied in wild plants. As many pathogens infest plants in the first days after germination, mycorrhiza-induced pathogen protection may be especially important in the first few weeks of plant establishment. Here, we investigated interacting effects of {AMF} and the seedling pathogen Pythium ultimum on the performance of six- to seven-week-old seedlings of six wild plant species of the family Asteraceae in a full factorial experiment. Plant species differed in their response to AMF, the pathogen and their interactions. {AMF} increased and the pathogen decreased plant biomass in one and three species, respectively. Two plant species were negatively affected by {AMF} in the absence, but positively or not affected in the presence of the pathogen, indicating protection by AMF. This mycorrhiza-induced pathogen protection is especially surprising as we could not detect mycorrhizal structure in the roots of any of the plants. Our results show that even seedlings without established intraradical hyphal network can profit from AMF, both in terms of growth promotion in the absence of a pathogen and pathogen protection. The function of {AMF} is highly species-specific, but tends to be similar for more closely related plant species, suggesting a phylogenetic component of mycorrhizal function. Further studies should test a wider range of plant species, as our study was restricted to one plant family, and investigate whether plants profit from early mycorrhizal benefits in the long term.

Initial vegetation, and spore density, diversity and evenness of different arbuscular mycorrhizal fungi morphotaxa 29,36 and 39 months after experimental vegetation removal, central Argentina

1. Dominant plant functional types (PFTs) are expected to be primary determinants of communities of other above- and below-ground organisms. Here, we report the effects of the experimental removal of different PFTs on arbuscular mycorrhizal fungi (AMF) communities in a shrubland ecosystem in central Argentina. 2. On the basis of the biomass-ratio hypothesis and plant resource use strategy theory, we expected the effect of removal of PFTs on AMF colonization and spores to be proportional to the biomass removed and to be stronger when more conservative PFTs were removed. The treatments applied were: undisturbed control (no plant removed), disturbed control (mechanical disturbance), no shrub (removal of deciduous shrubs), no perennial forb (removal of perennial forbs), no graminoid (removal of graminoids) and no annual forb (removal of annual forbs). AMF colonization was assessed after 5,17 and 29 months. Total density of AMF spores, richness and evenness of morpho-taxa, and AMF functional groups were quantified after 5,17,29,36 and 39 months. 3. Five months after the initial removal we found a significant reduction in total AMF colonization in all plots subjected to PFT removals and in the disturbed control plots, as compared with the undisturbed controls. This effect disappeared afterwards and no subsequent effect on total colonization and colonization by arbuscules was observed. In contrast, a significant increase in colonization by vesicles was observed in months 17 and 29, mainly in no graminoid plots. In general, treatments did not significantly affect AMF spores in the soil. On the other hand, no annual forb promoted transient (12-18 months) higher ammonia availability, and no shrub promoted lower nitrate availability in the longer term (24-28 months). 4. Synthesis. Our experiment, the first to investigate the effects of the removal of different PFTs on AMF communities in natural ecosystems, indicates that AMF communities are resilient to changes in the soil and in the functional composition of vegetation. Furthermore, it does not provide consistent evidence in support of the biomass-ratio hypothesis or differential trait-based direct or indirect effects of different PFTs on AMF in this particular system.

Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae.

Arbuscular mycorrhizal (AM) fungi (Order Glomales, Class Zygomycetes) are a diverse group of soil fungi that form mutualistic associations with the roots of most species of higher plants. Despite intensive study over the past 25 years, the phylogenetic relationships among AM fungi, and thus many details of evolution of the symbiosis, remain unclear. Cladistic analysis was performed on fatty acid methyl ester (FAME) profiles of 15 species in Gigaspora and Scutellospora (family Gigasporaceae) by using a restricted maximum likelihood approach of continuous character data. Results were compared to a parsimony analysis of spore morphological characters of the same species. Only one tree was generated from each character set. Morphological and developmental data suggest that species with the simplest spore types are ancestral whereas those with complicated inner wall structures are derived. Spores of those species having a complex wall structure pass through stages of development identical to the mature stages of simpler spores, suggesting a pattern of classical Haeckelian recapitulation in evolution of spore characters. Analysis of FAME profiles supported this hypothesis when Glomus leptotichum was used as the outgroup. However, when Glomus etunicatum was chosen as the outgroup, the polarity of the entire tree was reversed. Our results suggest that FAME profiles contain useful information and provide independent criteria for generating phylogenetic hypotheses in AM fungi. The maximum likelihood approach to analyzing FAME profiles also may prove useful for many other groups of organisms in which profiles are empirically shown to be stable and heritable.

The Effects of Arbuscular Mycorrhizal Fungi on four Legume Hosts in South Florida Pine Rockland Soils

This study addressed the effects of salinity and pot size on the interaction between leguminous plant hosts and arbuscular mycorrhizal fungi in four pine rockland soils using a shade house trap-plant experiment. Little is known about the belowground diversity of pine rocklands and the interactions between aboveground and belowground biota – an increased understanding of these interactions could lead to improved land management decisions, conservation and restoration efforts. Following twelve weeks of growth, plants were measured for root and shoot dry biomass and percent colonization by arbuscular mycorrhizal fungi. Overall, arbuscular mycorrhizal fungi had positive fitness effects on the four legume species (Cajanus cajan, Chamaecrista fasciculata, Tephrosia angustissima and Abrus precatorius), improving their growth rate, shoot and root biomass; pot size influenced plant-fungal interactions; and percent colonization by arbuscular mycorrhizal fungi was influenced by soil type as well as salinity.

Growth and symbiosis of plants with arbuscular mycorrhizal fungi in soil submitted to biochar application.

Arbuscular mycorrhizal fungi (AMF), which is intrinsically present or may be introduced in soils by inoculation, is an example of natural and renewable resource to increase plant nutrient uptake. This kind of fungi produces structures (hyphae, arbuscles and sometimes vesicles) inside the plant root cortex. This mutualistic relationship promotes plant gains in terms of water and nutrient absorption (mainly phosphorus). Biochar can benefit plant interaction with AMF, however, it can contain potentially toxic compounds such as heavy metals and organic compounds (e.g. dioxins, furans and polycyclic aromatic hydrocarbons), depending on the feedstock and pyrolysis conditions, which may damage organisms. For these reasons, the present work will approach the impacts of biochar application on soil attributes, AMF-plant symbiosis and its responses in plant growth and phosphorus uptake. Eucalyptus biochar produced at high temperatures increases sorghum growth; symbiosis with AMF; and enhances spore germination. Enhanced plant growth in the presence of high temperature biochar and AMF is a response of root branching stimulated by an additive effect between biochar characteristics and root colonization. Biochar obtained at low temperature reduces AMF spore germination; however it does not affect plant growth and symbiosis in soil.

大白花杜鹃菌根真菌多样性的研究

杜鹃花属(Rhododendron L.)植物分布广泛，研究发现所有的杜鹃花科植物都能形成一种特殊的菌根——杜鹃花类菌根(Ericoid Mycorrhiza)。杜鹃花类菌根对杜鹃花科植物在营养胁迫的环境下生长起到重要的作用。近几年，对杜鹃花类菌根的生物学和生态功能的研究越来越重视。我国是杜鹃花属植物资源最为丰富的国家，因此研究杜鹃花属植物菌根真菌多样性，充分利用杜鹃花特有的菌根资源，促进杜鹃花迁地保护成功具有重大的意义。 本研究以分布较广并且是中国特有的杜鹃花属植物——大白花杜鹃(Rhododendron decorum Franch.)的野生植株为研究对象，应用直接扩增根中真菌ITS区的分子鉴定方法和T-RFLP(末端限制性片段长度多态性)的分析方法，来研究其菌根真菌的多样性；并结合生态化学计量学特征分析、宿主遗传相似性及其群落组成分析等内容，探讨大白花杜鹃的菌根真菌－宿主植物－根际土壤三者之间的关系。主要结果如下： (1)通过用直接扩增真菌ITS区序列，揭示了大白花杜鹃根部真菌的多样性，本研究发现，野生大白杜鹃根部的真菌种类比较丰富，至少有26个ITS-taxa，包括子囊菌和担子菌共5个真菌目：Helotiales、Lecanorales(≡Agyriales)、Onygenales、Sebacinales和Thelephorales，其中包括典型的ERM真菌——树粉孢属Oidiodendron sp.(Myxotrichaceae)真菌。另外还发现了黑色有隔内生菌(Dark septate endophyte，DSE)以及一些未命名的子囊菌。担子菌在本研究中占有较大比例，尤其是蜡壳耳菌目真菌；此外还有较典型的外生菌根真菌——革菌目真菌。 (2)大白花杜鹃野生植株与栽培植株在菌根真菌种类组成上，有一定的相似性；在忽略种源差异等条件下相较而言，前者的物种丰富度远高于后者。 (3)大白花杜鹃菌根真菌多样性和丰富度同它的根际土壤与叶片的C、N、P含量以及C/N、N/P、土壤pH值、宿主的海拔高度等都没有显著的相关关系。 (4)在大白花杜鹃的菌根真菌群落组成方面，整体上保持了相当程度的相似性，同时还保持了一定水平的差异；大白杜鹃菌根真菌的种类是丰富的，优势度指数表明其多样性水平很高。 (5)大白花杜鹃的遗传距离与其菌根真菌群落组成结构有极显著的相关关系，宿主的种内遗传差异可能对菌根真菌群落物种组成产生选择偏好。 (6)大白花杜鹃的群落组成与其菌根真菌群落组成有极为显著的关联性，伴生种的菌根类型可能会影响宿主植物菌根真菌的物种组成结构。

Carbon availability affects nitrogen source utilisation by Hymenoscyphus ericae

We compared the ability of five strains of the ericoid mycorrhizal fungus Hymenoscyphus ericae to utilise glutamine, ammonium or nitrate at high or low carbon (C) availability. The pattern of intraspecific variation in growth was affected by C availability. When C supply was high, growth differences between strains were explained by the total amount of nitrogen (N) taken up, suggesting variation in uptake kinetics. Under C-limiting conditions, strain differences were linked with their nitrogen use efficiency, implying intraspecific differences in N metabolism. The relationship between growth on glutamine and pH shifts in the media indicated that there was intraspecific variation in glutamine transporters. In addition, the correlation between pH changes and the amount of glutamine-N recovered as ammonium in the media indicated that there were intraspecific variations within the enzymatic pathways involved in glutamine metabolism. Our findings, compared with those of a previous study involving the same ericoid strains, draw attention to the temporal variation in nitrogen source utilisation by ericoid mycorrhizal fungi when maintained in axenic culture.