Arbuscular Mycorrhizal Fungal Sequences Research Articles

Detection of rare variants among nuclei populating the arbuscular mycorrhizal fungal model species Rhizophagus irregularis DAOM197198.

Identifying genuine polymorphic variants is a significant challenge in sequence data analysis, although detecting low-frequency variants in sequence data is essential for estimating demographic parameters and investigating genetic processes, such as selection, within populations. Arbuscular mycorrhizal (AM) fungi are multinucleate organisms, in which individual nuclei collectively operate as a population, and the extent of genetic variation across nuclei has long been an area of scientific interest. In this study, we investigated the patterns of polymorphism discovery and the alternate allele frequency distribution by comparing polymorphism discovery in 2 distinct genomic sequence datasets of the AM fungus model species, Rhizophagus irregularis strain DAOM197198. The 2 datasets used in this study are publicly available and were generated either from pooled spores and hyphae or amplified single nuclei from a single spore. We also estimated the intraorganismal variation within the DAOM197198 strain. Our results showed that the 2 datasets exhibited different frequency patterns for discovered variants. The whole-organism dataset showed a distribution spanning low-, intermediate-, and high-frequency variants, whereas the single-nucleus dataset predominantly featured low-frequency variants with smaller proportions in intermediate and high frequencies. Furthermore, single nucleotide polymorphism density estimates within both the whole organism and individual nuclei confirmed the low intraorganismal variation of the DAOM197198 strain and that most variants are rare. Our study highlights the methodological challenges associated with detecting low-frequency variants in AM fungal whole-genome sequence data and demonstrates that alternate alleles can be reliably identified in single nuclei of AM fungi.

The influence of a soil amendment on the abundance and interaction of arbuscular mycorrhizal fungi with arable soils and host winter wheat.

Arbuscular mycorrhizal (AM) fungi have been shown to be associated with an estimated 70 % of vascular terrestrial plants. Such relationships have been shown to be sensitive to soil disturbance, for example, tillage in the preparation of a seed bed. From the application of arable soil management, AM fungal populations have been shown to be negatively impacted in abundance and diversity, reducing plant growth and development. The present study aims to utilise two sources (multipurpose compost and a commercial inocula) of mycorrhizal fungi for the amendment of arable soils supporting Zulu winter wheat under controlled conditions and quantify plant growth responses. A total of nine fields across three participating farms were sampled, each farm practicing either conventional, reduced, or zero tillage soil management exclusively. Soil textures were assessed for each sampled soil. Via the employment of AM fungal symbiosis quantification methods, AM fungi were compared between soil amendments and their effects on crop growth and development. The present study was able to quantify a mean 6 cm increase to crop height (P<0.001), 10 cm reduction to root length corresponding with a 2.45-fold increase in AM fungal arbuscular structures (P<0.001), a 1.15-fold increase in soil glomalin concentration corresponding to a 1.26-fold increase in soil carbon, and a 1.32-fold increase in the relative abundance of molecular identified AM fungal sequences for compost amended soils compared to control samples. Mycorrhizal inocula, however, saw no change to crop height or root length, AM fungal arbuscules were reduced by 1.43-fold, soil glomalin was additionally reduced by 1.55-fold corresponding to a reduction in soil carbon by 1.31-fold, and a reduction to relative AM fungal species abundance by 1.26-fold. The present study can conclude the addition of compost as an arable soil amendment is more beneficial for the restoration of AM fungi beneficial to wheat production and soil carbon compared to the addition of a commercial mycorrhizal inocula.

Fern species and seasonal variation alter arbuscular mycorrhizal fungal colonization and co-occurrence patterns in the Heishiding Natural Reserve, South China

The establishment of arbuscular mycorrhizal fungi (AMF) symbiosis is generally considered an important pathway that assists ferns in their adaption to a variable environment in the forest ecosystem. However, how both fern species and seasonal variation affect the symbiotic characteristics and AMF community structure remains largely unknown. In this study, the AMF colonization status of 19 fern species in the Heishiding Nature Reserve located in south China was assessed. The effects of seasons on AMF colonization and community structure were also determined. The results showed that both seasons and species significantly affected AMF colonization rate (1.88–56.03 %) and spore density (94–546/25 g dry soil). In addition, according to principal component analysis, in spring, AMF colonization and spore density differed considerably from other seasons. The total AMF sequences detected in spring were significantly higher than in winter (p < 0.05). Glomus was the dominant genus, accounting for >75 % of all sequences. The AMF Chao1 and Faith PD indexes of Blechnum orientale Linn as well as the Shannon and Simpson indexes of Selaginella doederleinii Hieron were significantly higher in spring than in winter (p < 0.05). According to partial least squares path modeling, season had a significant influence on AMF colonization by varying the AMF alpha diversity rather than their community composition. Notably, network analysis showed that seasonal variation induced differences in AMF network structure, keystone taxa, and the number of links between species. This study highlights the symbiotic relationship between ferns and AMF in different seasons. The results provide novel insights on terrestrial ecosystem management and fern conservation from the perspective of plant-AM symbiosis.

Effects of pavements on diversity and activity of mycorrhizal symbionts associated with urban trees

This study investigated the impact of different types of soil sealing on the communities of a group of beneficial plant symbionts, arbuscular mycorrhizal fungi (AMF), colonizing the roots of two shade trees, Celtis australis and Fraxinus ornus, frequently grown in urbanized sites. Such plants were grown in an experimental site, in northern Italy, established in November 2011 and subjected to four different pavement treatments: impermeable monolithic asphalt, permeable pavers, permeable concrete and unpaved soil. The diversity and composition of root AMF communities were assessed by PCR denaturating gradient gel electrophoresis of partial 18S rRNA gene, AMF taxa were identified by amplicon sequencing and mycorrhizal colonization was evaluated after root clearing and staining. For the first time, our molecular work revealed that impermeable pavements induced shifts in the composition of AMF communities associated to the roots of C. australis and F. ornus and impacted on the percentage of mycorrhizal root length. When the root-zone was covered with permeable pavements, a similar AMF community as that observed in the unpaved soil was detected, providing novel information to be utilised for reducing the disturbance caused by specific types of soil sealing on AMF symbionts, which play a key role in plant nutrition and health. A total of 45 AMF sequence types were detected, with Sclerocystis and Septoglomus as the most abundant phylotypes, accounting for 84% of the sequences. The predominance of Sclerocystis species in the roots of both tree species under impermeable pavements indicated their high and unforeseen tolerance towards harsh environmental conditions. Such species could be utilized as AMF inocula specifically selected for their proven resilience in paved sites, in order to exploit their ability to boost biogeochemical processes fundamental for energy fluxes and plant nutrition and health.

Isolation and identification of an arbuscular mycorrhizal fungus specifically associated with mycoheterotrophic seedlings of Gentiana zollingeri (Gentianaceae).

Gentiana zollingeri (Gentianaceae) is an initial mycoheterotrophic plant that depends on a specific group of arbuscular mycorrhizal (AM) fungi for carbon source during underground growth after seed germination. In this study, a mycorrhizal fungus dominant in mycoheterotrophic seedlings of G. zollingeri was successfully isolated from a soil core collected from a point close to a flowering G. zollingeri. The AM fungal isolate was identified as conspecific or closely related to Dominikia aurea (Glomeraceae) by spore morphology and molecular phylogeny. Basic Local Alignment Search Tool (BLAST) searches against the MaarjAM database showed that the nuclear small subunit ribosomal DNA sequences of the isolate matched the AM fungal sequences obtained from a wide range of plants in various ecosystems, including several mycoheterotrophs. Thus, it is suggested that the AM fungal isolate is one of the cheating susceptible AM fungi. Furthermore, the sequences corresponded to those of a group of AM fungi dominantly detected in Japanese temperate forests. Accordingly, there is a possibility that mycoheterotrophic plants, including seedlings of G. zollingeri, may target AM fungi with a wide host range and ubiquitous distribution.

Global taxonomic and phylogenetic assembly of AM fungi.

Arbuscular mycorrhizal (AM) fungi are a ubiquitous group of plant symbionts, yet processes underlying their global assembly - in particular the roles of dispersal limitation and historical drivers - remain poorly understood. Because earlier studies have reported niche conservatism in AM fungi, we hypothesized that variation in taxonomic community composition (i.e.,unweighted by taxon relatedness) should resemble variation in phylogenetic community composition (i.e., weighted by taxon relatedness) which reflects ancestral adaptations to historical habitat gradients. Because of the presumed strong dispersal ability of AM fungi, we also anticipated that the large-scale structure of AM fungal communities would track environmental conditions without regional discontinuity. We used recently published AM fungal sequence data (small-subunit ribosomal RNA gene) from soil samples collected worldwide to reconstruct global patterns in taxonomic and phylogenetic community variation. The taxonomic structure of AM fungal communities was primarily driven by habitat conditions, with limited regional differentiation, and there were two well-supported clusters of communities - occurring in cold and warm conditions. Phylogenetic structure was driven by the same factors, though all relationships were markedly weaker. This suggests that niche conservatism with respect to habitat associations is weakly expressed in AM fungal communities. We conclude that the composition of AM fungal communities tracks major climatic and edaphic gradients, with the effects of dispersal limitation and historic factors considerably less apparent than those of climate and soil.

Rhizophagus proliferus genome sequence reiterates conservation of genetic traits in AM fungi, but predicts higher saprotrophic activity.

Arbuscular mycorrhizal (AM) fungi are ubiquitous endosymbionts of terrestrial plants. It helps plants to extract more nutrients from the soil and enhances the plant tolerance to various ecological stress factors. The AM fungal genome sequence helps to identify the gene repertoires that are crucial for adaptation to different habitat and mechanisms for interaction with host plant. The present work comprises the first draft of the genome sequence of Rhizophagus proliferus, which is an important AM species present in biofertilizer consortia for agricultural purpose. The estimated genome size of R. proliferus is ~ 110 Mbps and its genomic assembly is 94.35% complete. Genome mining was carried out to identify putative gene families important for biological functions. A total of 22,526 protein-coding genes were estimated in the genome, with an abundance of kinases and reduced number of glycoside hydrolases as compared to other fungal classes. A striking finding in the R. proliferus genome was higher number of carbohydrate esterases (CE), which may suggest towards presence of higher saprotrophic activity in this species as compared to the previously reported AM fungi, which may indicate towards its role as a link between plants and soil mineral nutrients. The genome sequence and annotation of R. proliferus presented here would serve as an important reference for functional genomics studies required for developing biofertilizer formulations in future. In addition, the findings from this work may also prove important in deciphering molecular mechanisms in AM fungi that govern the host-specific interaction and associated agriculture benefits.

Ignored diversity of arbuscular mycorrhizal fungi in co-occurring mycotrophic and non-mycotrophic plants.

Arbuscular mycorrhizal fungi (AMF) colonization in roots of putative non-mycotrophic species has been known for decades, but our knowledge of AMF community structure in non-mycotrophic plants is limited. Here, we compared AMF species composition and diversity in roots of co-occurring mycotrophic and putative non-mycotrophic herbs in two wetlands. A SSU-ITS-LSU fragment in AMF rDNA was amplified, cloned and sequenced, and used to characterize the AMF community in the roots of 16 putative non-mycotrophic and 18 mycotrophic herbs. The results showed that AMF hyphae and vesicles, but not arbuscules, were commonly present in putative non-mycotrophic plants. A total of 971 AMF sequences were obtained, and these were finally assigned to 28 operational taxonomic units (OTUs). At both sites, AMF taxon richness and Hill number based on Shannon's index in the putative non-mycotrophic herbs were similar to those for mycotrophic plants, but AMF community composition between mycotrophic and non-mycotrophic plants was significantly different. Ten AMF OTUs were uniquely detected in the putative non-mycotrophic species, and two were identified as the AMF indicators in non-mycotrophic plants. These results implied that non-mycotrophic plants may harbor a potential source of AMF diversity previously ignored which should be included in our understanding of diversity, distribution pattern, and ecological significance of root-colonizing AMF. As the first direct comparison of AMF diversity and species composition between mycotrophic and putative non-mycotrophic species in wetlands, our study has important implications for the understanding of AMF distribution patterns.

SeSaMe: Metagenome Sequence Classification of Arbuscular Mycorrhizal Fungi-associated Microorganisms

Arbuscular mycorrhizal fungi (AMF) are plant root symbionts that play key roles in plant growth and soil fertility. They are obligate biotrophic fungi that form coenocytic multinucleated hyphae and spores. Numerous studies have shown that diverse microorganisms live on the surface of and inside their mycelia, resulting in a metagenome when whole-genome sequencing (WGS) data are obtained from sequencing AMF cultivated in vivo. The metagenome contains not only the AMF sequences, but also those from associated microorganisms. In this study, we introduce a novel bioinformatics program, Spore-associated Symbiotic Microbes (SeSaMe), designed for taxonomic classification of short sequences obtained by next-generation DNA sequencing. A genus-specific usage bias database was created based on amino acid usage and codon usage of a three consecutive codon DNA 9-mer encoding an amino acid trimer in a protein secondary structure. The program distinguishes between coding sequence (CDS) and non-CDS, and classifies a query sequence into a genus group out of 54 genera used as reference. The mean percentages of correct predictions of the CDS and the non-CDS test sets at the genus level were 71% and 50% for bacteria, 68% and 73% for fungi (excluding AMF), and 49% and 72% for AMF (Rhizophagus irregularis), respectively. SeSaMe provides not only a means for estimating taxonomic diversity and abundance but also the gene reservoir of the reference taxonomic groups associated with AMF. Therefore, it enables users to study the symbiotic roles of associated microorganisms. It can also be applicable to other microorganisms as well as soil metagenomes. SeSaMe is freely available at www.fungalsesame.org.

Communities of arbuscular mycorrhizal fungi in forest ecosystems in Japan's temperate region may be primarily constituted by limited fungal taxa.

We investigated arbuscular mycorrhizal (AM) fungal communities in secondary forests and/or Chamaecyparis obtusa plantations at eight study sites in Japan's temperate region. In the secondary forests, AM plants of the families Lauraceae, Sapindaceae, Rutaceae, Araliaceae, Rosaceae, Magnoliaceae, Cornaceae, Piperaceae, and Anacardiaceae were found. The AM fungal communities were evaluated based on compositions of the AM fungal operational taxonomic units (OTUs), which were clustered at a 97% similarity threshold of the sequences of a partial small subunit of a nuclear ribosomal RNA gene obtained from the plant roots. The compositions of AM fungal OTUs were significantly correlated with the plant family compositions and were significantly differentiated among the study sites and between the study forests. Interestingly, only 19 OTUs remained after selecting for those that had more than 1.0% of the total reads, and these 19 OTUs accounted for 86.3% of the total rarefied reads that were classified into 121 OTUs. Furthermore, three dominant OTUs constituted 48.0% of the total reads, and the most dominant OTU was found at all study sites, except at one. These results indicate that AM fungal communities are primarily constituted by limited AM fungal taxa in the forest ecosystems with diverse plant taxa in Japan's temperate region. The results of basic local algorithm search tool (BLAST) searches against MaarjAM, a database of AM fungal sequences, also revealed that the AM fungi which were the three dominant OTUs are distributed in forest ecosystems on a worldwide scale.

Evaluation of primer pairs for studying arbuscular mycorrhizal fungal community compositions using a MiSeq platform

Although many primers have been proposed for arbuscular mycorrhizal fungi (AMF)–specific sequencing, only a few studies have evaluated suitable primer pairs for MiSeq analysis. Here, we analyzed AMF community compositions using several primer pairs and evaluated which pair can detect a more diverse AMF community. Three AMF-specific primer pairs targeting the 18S and 28S rRNA genes and a fungal universal primer pair targeting the ITS region were examined using soil DNA as the template. The effect of nested PCR was also evaluated. Only a few AMF sequences were obtained from a fungal universal primer targeting the ITS region. On the other hand, use of AMF-specific primers improved AMF detection rates (35–98%). Particularly, almost all of the sequences obtained with the nested PCR were AMF-related sequences. However, the AMF community compositions were differentiated among the primer pairs. In addition, the number of AMF taxa and diversity decreased with the nested PCR, suggesting that it affected AMF community analysis.

The composition characteristics of arbuscular mycorrhizal fungal communities associated with barley in saline-alkaline soils in Central Anatolia

ABSTRACT Central Anatolia, which suffers from salinity, alkalinity, and drought stresses, is one of the most important cultivation regions of barley (Hordeum vulgare) in Turkey. Arbuscular mycorrhizal fungi (AMF) could promote barley production under several stresses; however, only a little information is available for AMF community composition in Turkish arable soils. In this study, barley root samples were collected from eight sites in the Central Anatolian region during the growing season (GS: April) and the harvest season (HS: July) in 2012, and the composition of AMF communities were elucidated based on the partial sequence of the AMF 18S rRNA gene using high-throughput sequencing technology. As a result, barley-AMF symbioses in this region were highly dominated by Glomeraceae (71.8% in GS and 59.2% in HS), followed by Claroideoglomeraceae (10.3% in GS and 15.9% in HS), Gigasporaceae (9.1% in GS and 13.1% in HS), and Acaulosporaceae (5.8% in GS and 7.7% in HS). Compared to Glomeraceae and Claroideoglomeraceae families, communities of Acaulosporaceae, Diversisporaceae, Paraglomeraceae, and Gigasporaceae consisted of fewer AMF species. The AMF evenness significantly increased from GS to HS. The most dominant AMF sequence, VTX00248 in the MaarjAM database, was closely related to Rhizophagus, which occupied 25.8% and 14.7% of the total AMF sequences in GS and HS, respectively. The relative abundance of AMF related to Rhizophagus tended to be reduced in HS, suggesting that the species could form mycorrhiza in the early stages of barley growth in this region. On the other hand, the relative abundance of Claroideoglomeraceae and Scutellosporaceae tended to increase in HS. Soil CaCO3 content significantly influenced AMF community compositions in GS, while soil pH and EC showed no significant impact on AMF community compositions. Based on discriminant analysis, 11 VTXs (related Acaulospora, Claroideoglomus, Funneliformis, Gigaspora, and Glomus) showed higher abundance in the barley roots grown in the soil with relatively high CaCO3 content, suggesting that these sequences might be adapted to such an environment.

Salt is a main factor shaping community composition of arbuscular mycorrhizal fungi along a vegetation successional series in the Yellow River Delta

In coastal wetlands, salinity significantly influences the vegetation successional series. Arbuscular mycorrhizal fungal (AMF) are ubiquitous soil microorganisms which play important roles in plant establishment, diversifying plant communities and accelerating successional progress by improving the soil health. However, the structure and function of AMF communities in coastal wetlands are poorly understood. Here we investigated the AMF communities along a vegetation successional series (from intertidal to supratidal flats) and the sediment physicochemical factors affecting the variations. AMF community composition and diversity were analyzed by 18S rRNA genes using high-throughput sequencing technology. The results revealed that the maximum number of AMF sequences belonged to Glomus 74,660, 62.75%. The relative abundance of the top three genera differed significantly along the vegetation successional series. Redundancy analysis (RDA) results demonstrated that salinity was a major environmental factor structuring the AMF community in the sediment along the vegetation successional series. No significant differences of AMF diversity were observed between different sediment layers. Additionally, total nitrogen (TN) and total carbon (TC), important soil nutrients in young emergent wetland ecosystems, also contributed to AMF community structure. The results of this study provide a better understanding of the AMF community and the major factors impacting it along a coastal wetland successional series composed of different vegetation types.

Racocetra crispa (Glomeromycotina) delimited by integrative evidence based on morphology, long continuous nuclear rDNA sequencing and phylogeny

Here, we describe a new ornamented arbuscular mycorrhizal (AM) fungus, Racocetra crispa sp. nov. isolated from maize fields from the central region of Minas Gerais State, Brazil. For the first time, a Glomeromycotina species is described using a long continuous nuclear rDNA sequence fragment, which encompasses the nearly complete 18S SSU sequence gene until the 3′ end of the D2 region of the 28S LSU (~ 3100 bp), which allows for comparison with sequences obtained from regions used for fungal metagenomic studies, species description, and AM fungi DNA-barcode. The new species forms dark brown to black spores, approx. 340–510 μm on in diam., on sporogenous cells. The spores have unique “cloud/flower” projections on the spore surface, two walls, and differentiate a multiple-lobed germ shield with up to 8–12 germ tube initiations. The analysis of the intra- and interspecific DNA-barcode sequence variation within the Racocetra showed that the intragenomic polymorphism among the clones of R. crispa (0–2 %) is within the lower range for the genus. The V3–V4 region of the SSU nrDNA has no resolution to discriminate Racocetra at species level, but from this fragment, we found homology between R. crispa and environmental sequences from two metagenomics studies, one carried out in Brazil at the fungus type location and the other in New Zealand. The integration between AM fungal sequences from reference strains and those obtained from environmental sequences in Glomeromycotina is still a problematic issue, mainly due to the reduced number of AM fungal species characterized based on DNA sequences.

Arbuscular mycorrhizal fungi in two vertical-flow wetlands constructed for heavy metal-contaminated wastewater bioremediation.

Over the last three decades, the presence of arbuscular mycorrhizal fungi (AMF) in wetland habitats had been proven, and their roles played in wetland ecosystems and potential functions in wastewater bioremediation technical installations are interesting issues. To increase knowledge on the functions of AMF in the plant-based bioremediation of wastewater, we constructed two vertical-flow wetlands planting with Phragmites australis and investigated AMF distribution in plant roots and their roles played in purification of wastewater polluted by heavy metals (HMs), utilizing the Illumina sequencing technique. A total of 17 operational taxonomic units (OTUs) from 33,031 AMF sequences were obtained, with Glomus being the most dominant. P. australis living in the two vertical-flow constructed wetlands (CWs) harbored diverse AMF comparable with the AM fungal communities in upland habitats. The AMF composition profiles of CW1 (vegetated with non-inoculated plants) and CW2 (vegetated with mycorrhizal plants inoculated with Rhizophagus intraradices) were significantly different. CW1 (15 OTUs) harbored more diverse AMF than CW2 (7 OTUs); however, CW2 harbored much more OTU13 than CW1. In addition, a zipf species abundance distribution (SAD), which might due to the heavy overdominance of OTU13, was observed across AM fugal taxa in P. australis roots of the two CWs. CW1 and CW2 showed high (> 70%) removal capacity of HMs. CW2 exhibited significant higher Cd and Zn removal efficiencies than CW1 (CK) (p = 0.005 and p = 0.008, respectively). It was considered that AMF might play a role in HM removal in CWs.

Arbuscular Mycorrhizal Fungal Community Composition in Carludovica palmata, Costus scaber and Euterpe precatoria from Weathered Oil Ponds in the Ecuadorian Amazon.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous to most natural and anthropized ecosystems, and are often found in polluted environments. However, their occurrence and community composition in highly weathered petroleum-polluted soils has been infrequently reported. In the present study, two ponds of weathered crude oil and their surrounding soil from the Charapa field in the Amazon region of Ecuador were selected and root colonization by AMF of their native plants investigated. The AMF community was further analyzed in three selected plant species (i.e., Carludovica palmata, Costus scaber and Euterpe precatoria) present in the two ponds and the surrounding soil. A fragment covering partial SSU, the whole ITS and partial LSU rDNA region was amplified (i.e., 1.5 kb), cloned and sequenced from the roots of each host species. AMF root colonization exceeded 56% in all plant species examined and no significant difference was observed between sites or plants. For AMF community analysis, a total of 138 AMF sequences were obtained and sorted into 32 OTUs based on clustering (threshold ≥97%) by OPTSIL. The found OTUs belonged to the genera Rhizophagus (22%), Glomus (31%), Acaulospora (25%) and Archaeospora (22%). Glomus and Archaeospora were always present regardless of the plant species or the site. Acaulospora was found in the three plant species and in the two ponds while Rhizophagus was revealed only in the surrounding soil in one plant species (Euterpe precatoria). Our study contributed to the molecular community composition of AMF and revealed an unexpected high presence of four AMF genera which have established a symbiosis with roots of native plants from the Amazon forest under high polluted soil conditions.

Communities of arbuscular mycorrhizal fungi under Picconia azorica in native forests of Azores

Arbuscular mycorrhizal fungi (AMF) from the rhizosphere of the endemic Laurisilva tree, Picconia azorica, were characterised at two sites in each of two Azorean islands (Terceira and Sao Miguel). Forty-six spore morphotypes were found, and DNA extraction was attempted from individual spores of each of these. DNA was obtained from 18 of the morphotypes, from which a 1.5 kb long fragment of the nuclear ribosomal RNA gene (SSU-ITS-LSU) was sequenced. A total of 125 AMF sequences were obtained and assigned to 18 phylotypes. Phylogenetic analysis revealed sequences belonging to the families, Acaulosporaceae, Archaeosporaceae, Claroideoglomeraceae, Gigasporaceae and Glomeraceae. Phylotype richness changed between islands and between sampling sites at both islands suggesting that geographical and historical factors are determinant in shaping AMF communities in native forest of Azores. Ecological analysis of the molecular data revealed differences in AMF community composition between islands. In Terceira, the rhizosphere of P. azorica was dominated by species belonging to Acaulosporaceae and Glomeraceae, while Sao Miguel was dominated by members of Glomeraceae and Gigasporaceae. This is the first molecular study of AMF associated with P. azorica in native forest of the Azores. These symbiont fungi are key components of the ecosystem. Further research is needed to develop their use as promoters of plant establishment in conservation and restoration of such sites.

Molecular Diversity of Arbuscular Mycorrhizae in Roots of Juniperus virginiana Invasive to Grasslands

Core Ideas Glomus spp. dominate the arbuscular mycorrhizal fungal (AMF) community in eastern red cedar (ERC) roots. Young seedlings harbor the most diverse AMF, similar to grassland soils from the same site. AMF diversity within ERC roots tended to decrease with plant age suggesting narrower plant and edaphic selection pressures with increasing ERC stand density. A change in biotic and abiotic factors resulting from displacement of grassland with ERC is likely responsible for successional changes in the AMF community. Eastern red cedar (ERC; Juniperus virginiana) is an invasive species of juniper native to eastern North America that causes significant economic loss to grasslands. Recent findings of high molecular diversity of arbuscular mycorrhizal fungi (AMF) in soils under ERC stands led us to consider that the invasiveness of ERC into grasslands may arise in part from its ability to form associations with extant grassland AMF communities. The diversity of AMF in roots of ERC was determined for five age groups ranging from 1 to 65 yr old and varying in stand density from individual seedlings in grassland (&lt;5 yr) to closed ERC canopies devoid of grass species (65 yr). Roots were analyzed by nested polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) fingerprinting, cloning, and insert screening by DGGE, and sequencing of ribosomal DNA small‐subunit regions. Phylogenetically analyzed AMF sequences were used to define phylotypes. The majority of the sequences obtained were classified into Glomus Group A, with only a few belonging to Gigasporaceae and Acaulosporaceae, the latter two largely restricted to roots from ERC &lt; 5 yr old. The richness of phylotypes tended to decrease with time, ranging from 11 in the youngest age group (1–5 yr) to 4 in the oldest age group (46–65 yr). Abundance of phylotypes varied greatly among the age groups. Overall, AMF diversity within ERC roots tended to decrease with plant age suggesting narrower plant and edaphic selection pressures with increasing ERC stand density.

Changes in the composition of native root arbuscular mycorrhizal fungal communities during a short-term cover crop-maize succession

Arbuscular mycorrhizal fungi (AMF) establish mutualistic associations with the most important agricultural food and feed crops, sustaining plant growth, nutrient uptake and tolerance of biotic and abiotic stresses. Scanty information is available on the role played by crop identity and diversity as a driving force shaping AMF species communities in the field, in particular in low-input and organic farming, where crop rotation and the use of cover crops are common practices. Here, using a molecular approach, we investigated whether plant communities established in low and high diversity cover crop treatments affect the composition of native AMF root communities of subsequent maize in a Mediterranean organic agroecosystem. A total of 16 AMF sequence types were detected, with Acaulospora cavernata as the most abundant phylotype, accounting for 37.4 % of the sequences, followed by Funneliformis mosseae, Claroideoglomus lamellosum and Rhizoglomus intraradices. Sequences matching to Funneliformis caledonium, Diversispora aurantia, Diversispora epigaea and Archaeospora schenckii corresponded to less than 2.0 % of the total. The most abundant sequences retrieved in plants from cover crop treatments were represented by A. cavernata, while sequences in maize roots were related to F. mosseae, R. intraradices and Glomus sp. Such data show for the first time a change in the composition of native AMF communities colonizing maize roots, which was independent of the identity and diversity of the preceding crop. Our findings suggest that host preference may represent a strong driver of AMF community dynamics in agroecosystems, differentially boosting or depressing AMF species, possibly in relation to their functional significance.

Distribution of Petrosavia sakuraii (Petrosaviaceae), a rare mycoheterotrophic plant, may be determined by the abundance of its mycobionts.

Petrosavia sakuraii (Petrosaviaceae) is a rare, mycoheterotrophic plant species that has a specific symbiotic interaction with a narrow clade of arbuscular mycorrhizal (AM) fungi. In the present study, we tested the hypothesis that the distribution and abundance of mycobionts in two P. sakuraii habitats, Nagiso and Sengenyama (central Honshu, Japan), determine the distribution pattern of this rare plant. Nagiso is a thriving habitat with hundreds of P. sakuraii individuals per 100m(2), whereas Sengenyama is a sparsely populated habitat with fewer than 10 individuals per 100m(2). AM fungal communities associated with tree roots were compared at 20-cm distances from P. sakuraii shoots between the two habitats by molecular identification of AM fungal partial sequences of the small subunit ribosomal RNA gene. The percentage of AM fungal sequences showing over 99% identity with those of the dominant P. sakuraii mycobionts was high (54.9%) in Nagiso, but low (13.2%) in Sengenyama. Accordingly, the abundance of P. sakuraii seems to reflect the proportion of potential mycobionts. It is likely that P. sakuraii mycobionts are not rare in Japanese warm temperate forests since 11.2% of AM fungal sequences previously obtained from a deciduous broad-leaved forest devoid of P. sakuraii in Mizuho, central Honshu, Japan, were >99% identical to those of the dominant P. sakuraii mycobionts. Thus, results suggest that the abundant mycobionts may be required for sufficient propagation of P. sakuraii, and this quantitative trait of AM fungal communities required for P. sakuraii may explain the rarity of this plant.