Increased sequencing depth does not increase captured diversity of arbuscular mycorrhizal fungi.

The arrival of 454 sequencing represented a major breakthrough by allowing deeper sequencing of environmental samples than was possible with existing Sanger approaches. Illumina MiSeq provides a further increase in sequencing depth but shorter read length compared with 454 sequencing. We explored whether Illumina sequencing improves estimates of arbuscular mycorrhizal (AM) fungal richness in plant root samples, compared with 454 sequencing. We identified AM fungi in root samples by sequencing amplicons of the SSU rRNA gene with 454 and Illumina MiSeq paired-end sequencing. In addition, we sequenced metagenomic DNA without prior PCR amplification. Amplicon-based Illumina sequencing yielded two orders of magnitude higher sequencing depth per sample than 454 sequencing. Initial analysis with minimal quality control recorded five times higher AM fungal richness per sample with Illumina sequencing. Additional quality control of Illumina samples, including restriction of the marker region to the most variable amplicon fragment, revealed AM fungal richness values close to those produced by 454 sequencing. Furthermore, AM fungal richness estimates were not correlated with sequencing depth between 300 and 30,000 reads per sample, suggesting that the lower end of this range is sufficient for adequate description of AM fungal communities. By contrast, metagenomic Illumina sequencing yielded very few AM fungal reads and taxa and was dominated by plant DNA, suggesting that AM fungal DNA is present at prohibitively low abundance in colonised root samples. In conclusion, Illumina MiSeq sequencing yielded higher sequencing depth, but similar richness of AM fungi in root samples, compared with 454 sequencing.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

A genetic discontinuity in root-nodulating bacteria of cultivated pea in the Indian trans-Himalayas.

Evolutionary relationships of 120 root-nodulating bacteria isolated from the nodules of Pisum sativum cultivated at 22 different locations of the trans-Himalayan valleys of Lahaul and Spiti in the state of Himachal Pradesh of India were studied using 16S rRNA gene PCR-RFLP, ERIC-PCR, sequencing of 16S rRNA, atpD, recA, nodC and nifH genes, carbon-source utilization pattern (BIOLOG™), and whole-cell fatty acid profiling. The results demonstrated that all isolates belonged to Rhizobium leguminosarum symbiovar viciae (Rlv). Isolates from the two valleys were clearly separated on the basis of ERIC fingerprints, carbon-source utilization pattern, and whole-cell fatty acid methyl esters. Phylogenetic analysis of atpD, recA, nodC and nifH genes revealed a common Rlv sublineage in Spiti valley. Lahaul valley isolates were represented by three sequence types of atpD and recA genes, and four sequence types of nodC and nifH genes. Genotypes from the two valleys were completely distinct, except for two Lahaul isolates that shared nodC and nifH sequences with Spiti isolates but were otherwise more similar to other Lahaul isolates. Isolates from the two highest Spiti valley sites (above 4000 m) had a distinctive whole-cell fatty acid profile. Spiti valley isolates are closely related to Rlv sublineages from Xinjiang and Shanxi provinces in China, while Lahaul valley isolates resemble cosmopolitan strains of the western world. The high mountain pass between these valleys represents a boundary between two distinct microbial populations.

Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other alpha-Proteobacteria in cucumber and tomato crops affected by root mat.

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring alpha-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium: An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring alpha-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).

Molecular diversity of Frankia in root nodules of Alnus incana grown with inoculum from polluted urban soils.

ABSTRACT The establishment and growth of trees can be compromised by soil contamination which can reduce populations of key microbial symbionts. We describe the colonisation of grey alder (Alnus incana) by Frankia from 10 urban soils with varying degrees of organic and inorganic pollution. Principal components analysis (PCA) of soil chemical profiles showed a separation of remediated and unremediated soils. A. incana seedlings were used as trap plants to capture the microsymbiont from soil. After 6 months growth, nodulation was lowest on trees grown with the most contaminated soils. Plant biomass was positively correlated with root nodule biomass and negatively correlated with PAH concentration. DNA was isolated from nodules for the analysis of Frankia genetic diversity. The polymerase chain reaction (PCR) was used to amplify the 16S-23S intergenic spacer (IGS) of Frankia ribosomal DNA. PCR products were subject to restriction digestion yielding 10 restriction fragment length polymorphism (RFLP) types from 72 nodules analysed. Our results demonstrate that each soil supports a distinct nodulating Frankia community. Partial 16S sequencing placed most strains in Frankia clusters 1a and 1b, which are typically Alnus-infecting, but sequences from several nodules obtained from a gasworks soil belonged to cluster 3, normally associated with Elaeagnus. These results show for the first time that polluted soils can be an effective source of Alnus-infective Frankia. Inoculation with site-adapted Frankia under greenhouse conditions could thus be an appropriate strategy to increase the symbiotic capacity of A. incana and to improve its chances of survival and growth when planted on polluted soils.

Co-existing grass species have distinctive arbuscular mycorrhizal communities.

Arbuscular mycorrhizal (AM) fungi are biotrophic symbionts colonizing the majority of land plants, and are of major importance in plant nutrient supply. Their diversity is suggested to be an important determinant of plant community structure, but the influence of host-plant and environmental factors on AM fungal community in plant roots is poorly documented. Using the terminal restriction fragment length polymorphism (T-RFLP) strategy, the diversity of AM fungi was assessed in 89 roots of three grass species (Agrostis capillaris, Festuca rubra, Poa pratensis) that co-occurred in the same plots of a field experiment. The impact of different soil amendments (nitrogen, lime, nitrogen and lime) and insecticide application on AM fungal community was also studied. The level of diversity found in AM fungal communities using the T-RFLP strategy was consistent with previous studies based on clone libraries. Our results clearly confirm that an AM fungal host-plant preference exists, even between different grass species. AM communities colonizing A. capillaris were statistically different from the others (P < 0.05). Although grass species evenness changed in amended soils, AM fungal community composition in roots of a given grass species remained stable. Conversely, in plots where insecticide was applied, we found higher AM fungal diversity and, in F. rubra roots, a statistically different AM fungal community.

Symbiotic and genetic diversity of Rhizobium galegae isolates collected from the Galega orientalis gene center in the Caucasus.

This paper explores the relationship between the genetic diversity of rhizobia and the morphological diversity of their plant hosts. Rhizobium galegae strains were isolated from nodules of wild Galega orientalis and Galega officinalis in the Caucasus, the center of origin for G. orientalis. All 101 isolates were characterized by genomic amplified fragment length polymorphism fingerprinting and by PCR-restriction fragment length polymorphism (RFLP) of the rRNA intergenic spacer and of five parts of the symbiotic region adjacent to nod box sequences. By all criteria, the R. galegae bv. officinalis and R. galegae bv. orientalis strains form distinct clusters. The nod box regions are highly conserved among strains belonging to each of the two biovars but differ structurally to various degrees between the biovars. The findings suggest varying evolutionary pressures in different parts of the symbiotic genome of closely related R. galegae biovars. Sixteen R. galegae bv. orientalis strains harbored copies of the same insertion sequence element; all were isolated from a particular site and belonged to a limited range of chromosomal genotypes. In all analyses, the Caucasian R. galegae bv. orientalis strains were more diverse than R. galegae bv. officinalis strains, in accordance with the gene center theory.

Molecular diversity of arbuscular mycorrhizal fungi and patterns of host association over time and space in a tropical forest.

We have used molecular techniques to investigate the diversity and distribution of the arbuscular mycorrhizal (AM) fungi colonizing tree seedling roots in the tropical forest on Barro Colorado Island (BCI), Republic of Panama. In the first year, we sampled newly emergent seedlings of the understory treelet Faramea occidentalis and the canopy emergent Tetragastris panamensis, from mixed seedling carpets at each of two sites. The following year we sampled surviving seedlings from these cohorts. The roots of 48 plants were analysed using AM fungal-specific primers to amplify and clone partial small subunit (SSU) ribosomal RNA gene sequences. Over 1300 clones were screened for random fragment length polymorphism (RFLP) variation and 7% of these were sequenced. Compared with AM fungal communities sampled from temperate habitats using the same method, the overall diversity was high, with a total of 30 AM fungal types identified. Seventeen of these types have not been recorded previously, with the remainder being similar to types reported from temperate habitats. The tropical mycorrhizal population showed significant spatial heterogeneity and nonrandom associations with the different hosts. Moreover there was a strong shift in the mycorrhizal communities over time. AM fungal types that were dominant in the newly germinated seedlings were almost entirely replaced by previously rare types in the surviving seedlings the following year. The high diversity and huge variation detected across time points, sites and hosts, implies that the AM fungal types are ecologically distinct and thus may have the potential to influence recruitment and host composition in tropical forests.

Arbuscular mycorrhizal community composition associated with two plant species in a grassland ecosystem.

Arbuscular mycorrhizal (AM) fungi are biotrophic symbionts colonizing about two-thirds of land plant species and found in all ecosystems. They are of major importance in plant nutrient supply and their diversity is suggested to be an important determinant of plant community composition. The diversity of the AM fungal community composition in the roots of two plant species (Agrostis capillaris and Trifolium repens) that co-occurred in the same grassland ecosystem was characterized using molecular techniques. We analysed the small subunit (SSU) ribosomal RNA gene amplified from a total root DNA extract using AM fungal-specific primers. A total of 2001 cloned fragments from 47 root samples obtained on four dates were analysed by restriction fragment length polymorphism, and 121 of them were sequenced. The diversity found was high: a total of 24 different phylotypes (groups of phylogenetically related sequences) colonized the roots of the two host species. Phylogenetic analyses demonstrate that 19 of these phylotypes belonged to the Glomaceae, three to the Acaulosporaceae and two to the Gigasporaceae. Our study reveals clearly that the AM fungal community colonizing T. repens differed from that colonizing A. capillaris, providing evidence for AM fungal host preference. In addition, our results reveal dynamic changes in the AM fungal community through time.

Molecular diversity of arbuscular mycorrhizal fungi colonising arable crops.

We used differences in small subunit ribosomal RNA genes to identify groups of arbuscular mycorrhizal fungi that are active in the colonisation of plant roots growing in arable fields around North Yorkshire, UK. Root samples were collected from four arable fields and four crop species, fungal sequences were amplified from individual plants by the polymerase chain reaction using primers NS31 and AM1. The products were cloned and 303 clones were classified by their restriction pattern with HinfI or RsaI; 72 were subsequently sequenced. Colonisation was dominated by Glomus species with a preponderance of only two sequence types, which are closely related. There is evidence for seasonal variation in colonisation in terms of both level of colonisation and sequence types present. Fungal diversity was much lower than that previously reported for a nearby woodland.