Taxon-specific PCR primers to detect two inconspicuous arbuscular mycorrhizal fungi from temperate agricultural grassland

Taxon-specific polymerase chain reaction (PCR) primers enable detection of arbuscular mycorrhizal fungi (AMF, Glomeromycota) in plant roots where the fungi lack discriminative morphological and biochemical characters. We designed and validated pairs of new PCR primers targeted to the flanking regions of the variable domain 1 of the nuclear ribosomal large subunit RNA gene to specifically detect Acaulospora paulinae and an undescribed member of the Diversisporaceae. These two fungal taxa, sporulating late in soil-trap cultures and showing small, faintly coloured spores and weakly staining intraradical structures, were frequently found in roots of Trifolium repens from a high-input agricultural grassland. The newly developed PCR primers may thus enable studies on two inconspicuous AMF taxa that appear to have been overlooked in previous molecular AMF community analyses and for which no specific PCR primers have been published.     

Flooding greatly affects the diversity of arbuscular mycorrhizal fungi communities in the roots of wetland plants

The communities of arbuscular mycorrhizal fungi (AMF) colonizing the roots of three mangrove species were characterized along a tidal gradient in a mangrove swamp. A fragment, designated SSU-ITS-LSU, including part of the small subunit (SSU), the entire internal transcribed spacer (ITS) and part of the large subunit (LSU) of rDNA from samples of AMF-colonized roots was amplified, cloned and sequenced using AMF-specific primers. Similar levels of AMF diversity to those observed in terrestrial ecosystems were detected in the roots, indicating that the communities of AMF in wetland ecosystems are not necessarily low in diversity. In total, 761 Glomeromycota sequences were obtained, which grouped, according to phylogenetic analysis using the SSU-ITS-LSU fragment, into 23 phylotypes, 22 of which belonged to Glomeraceae and one to Acaulosporaceae. The results indicate that flooding plays an important role in AMF diversity, and its effects appear to depend on the degree (duration) of flooding. Both host species and tide level affected community structure of AMF, indicating the presence of habitat and host species preferences.     

Combining nested PCR and restriction digest of the internal transcribed spacer region to characterize arbuscular mycorrhizal fungi on roots from the field

Identification of arbuscular mycorrhizal fungi (AMF) on roots is almost impossible with morphological methods and, due to the presence of contaminating fungi, it is also difficult with molecular biological techniques. To allow broad investigation of the population structure of AMF in the field, we have established a new method to selectively amplify the internal transcribed spacer (ITS) region of most AMF with a unique primer set. Based on available sequences of the rDNA, one primer pair specific for AMF and a few other fungal groups was designed and combined in a nested PCR with the already established primer pair ITS5/ITS4. Amplification from contaminating organisms was reduced by an AluI restriction after the first reaction of the nested PCR. The method was assessed at five different field sites representing different types of habitats. Members of all major groups within the Glomeromycota (except Archaeosporaceae) were detected at the different sites. Gigasporaceae also proved detectable with the method based on cultivated strains.     

Real-time PCR and microscopy: are the two methods measuring the same unit of arbuscular mycorrhizal fungal abundance?

To enable quantification of mycelial abundance in mixed-species environments, eight new TaqMan((R)) real-time PCR assays were developed for five arbuscular mycorrhizal fungal (AMF, Glomeromycota) taxa. The assays targeted genes encoding 18S rRNA or actin, and were tested on DNA from cloned gene fragments, from spores, mycelia, and from root-free soil, and on reverse-transcribed rRNA templates from entire mycelia and from colonized roots. The assays showed high specificity, sensitivity, and reproducibility, enabling reliable quantitation over broad ranges of template molecules. From cultured mycelia, DNA and RNA measures both correlated with spore number rather than extraradical hyphal length, and epifluorescence microscopy identified pronounced heterogeneity in vitality and nuclear distribution in hyphae. Root colonization was also spatially heterogeneous, as shown by a mixing experiment with root fragments of different length. Therefore, although real-time PCR can reproducibly and accurately quantify AMF nucleic acids, these are poorly correlated with visual measures because of spatial heterogeneity.     

Global sampling of plant roots expands the described molecular diversity of arbuscular mycorrhizal fungi

We aimed to enhance understanding of the molecular diversity of arbuscular mycorrhizal fungi (AMF) by building a new global dataset targeting previously unstudied geographical areas. In total, we sampled 96 plant species from 25 sites that encompassed all continents except Antarctica. AMF in plant roots were detected by sequencing the nuclear SSU rRNA gene fragment using either cloning followed by Sanger sequencing or 454-sequencing. A total of 204 AMF phylogroups (virtual taxa, VT) were recorded, increasing the described number of Glomeromycota VT from 308 to 341 globally. Novel VT were detected from 21 sites; three novel but nevertheless widespread VT (Glomus spp. MO-G52, MO-G53, MO-G57) were recorded from six continents. The largest increases in regional VT number were recorded in previously little-studied Oceania and in the boreal and polar climatic zones — this study providing the first molecular data from the latter. Ordination revealed differences in AM fungal communities between different continents and climatic zones, suggesting that both biogeographic history and environmental conditions underlie the global variation of those communities. Our results show that a considerable proportion of Glomeromycota diversity has been recorded in many regions, though further large increases in richness can be expected in remaining unstudied areas.     

The Largest Subunit of RNA Polymerase II as a New Marker Gene to Study Assemblages of Arbuscular Mycorrhizal Fungi in the Field

Due to the potential of arbuscular mycorrhizal fungi (AMF, Glomeromycota) to improve plant growth and soil quality, the influence of agricultural practice on their diversity continues to be an important research question. Up to now studies of community diversity in AMF have exclusively been based on nuclear ribosomal gene regions, which in AMF show high intra-organism polymorphism, seriously complicating interpretation of these data. We designed specific PCR primers for 454 sequencing of a region of the largest subunit of RNA polymerase II gene, and established a new reference dataset comprising all major AMF lineages. This gene is known to be monomorphic within fungal isolates but shows an excellent barcode gap between species. We designed a primer set to amplify all known lineages of AMF and demonstrated its applicability in combination with high-throughput sequencing in a long-term tillage experiment. The PCR primers showed a specificity of 99.94% for glomeromycotan sequences. We found evidence of significant shifts of the AMF communities caused by soil management and showed that tillage effects on different AMF taxa are clearly more complex than previously thought. The high resolving power of high-throughput sequencing highlights the need for quantitative measurements to efficiently detect these effects.     

Arbuscular mycorrhizal fungi (Glomeromycota) harbour ancient fungal tubulin genes that resemble those of the chytrids (Chytridiomycota)

The genes encoding alpha- and beta-tubulins have been widely sampled in most major fungal phyla and they are useful tools for fungal phylogeny. Here, we report the first isolation of alpha-tubulin sequences from arbuscular mycorrhizal fungi (AMF). In parallel, AMF beta-tubulins were sampled and analysed to identify the presence of paralogs of this gene. The AMF alpha-tubulin amino acid phylogeny was congruent with the results previously reported for AMF beta-tubulins and showed that AMF tubulins group together at a basal position in the fungal clade and showed high sequence similarities with members of the Chytridiomycota. This is in contrast with phylogenies for other regions of the AMF genome. The amount and nature of substitutions are consistent with an ancient divergence of both orthologs and paralogs of AMF tubulins. At the amino acid level, however, AMF tubulins have hardly evolved from those of the chytrids. This is remarkable given that these two groups are ancient and the monophyletic Glomeromycota probably diverged from basal fungal ancestors at least 500 million years ago. The specific primers we designed for the AMF tubulins, together with the high molecular variation we found among the AMF species we analysed, make AMF tubulin sequences potentially useful for AMF identification purposes.     

Disclosing arbuscular mycorrhizal fungal biodiversity in soil through a land-use gradient using a pyrosequencing approach

The biodiversity of arbuscular mycorrhizal fungi (AMF) communities present in five Sardinian soils (Italy) subjected to different land-use (tilled vineyard, covered vineyard, pasture, managed meadow and cork-oak formation) was analysed using a pyrosequencing-based approach for the first time. Two regions of the 18S ribosomal RNA gene were considered as molecular target. The pyrosequencing produced a total of 10924 sequences: 6799 from the first and 4125 from the second target region. Among these sequences, 3189 and 1003 were selected to generate operational taxonomic units (OTUs) and to evaluate the AMF community richness and similarity: 117 (37 of which were singletons) and 28 (nine of which were singletons) unique AMF OTUs were detected respectively. Within the Glomeromycota OTUs, those belonging to the Glomerales order were dominant in all the soils. Diversisporales OTUs were always detected, even though less frequently, while Archaeosporales and Paraglomerales OTUs were exclusive of the pasture soil. Eleven OTUs were shared by all the soils, but each of the five AMF communities showed particular features, suggesting a meaningful dissimilarity among the Glomeromycota populations. The environments with low inputs (pasture and covered vineyard) showed a higher AMF biodiversity than those subjected to human input (managed meadow and tilled vineyard). A reduction in AMF was found in the cork-oak formation because other mycorrhizal fungal species, more likely associated to trees and shrubs, were detected. These findings reinforce the view that AMF biodiversity is influenced by both human input and ecological traits, illustrating a gradient of AMF communities which mirror the land-use gradient. The high number of sequences obtained by the pyrosequencing strategy has provided detailed information on the soil AMF assemblages, thus offering a source of light to shine on this crucial soil microbial group.     

Evidence of differences between the communities of arbuscular mycorrhizal fungi colonizing galls and roots of Prunus persica infected by the root-knot nematode Meloidogyne incognita

Arbuscular mycorrhizal fungi (AMF) play important roles as plant protection agents, reducing or suppressing nematode colonization. However, it has never been investigated whether the galls produced in roots by nematode infection are colonized by AMF. This study tested whether galls produced by Meloidogyne incognita infection in Prunus persica roots are colonized by AMF. We also determined the changes in AMF composition and biodiversity mediated by infection with this root-knot nematode. DNA from galls and roots of plants infected by M. incognita and from roots of noninfected plants was extracted, amplified, cloned, and sequenced using AMF-specific primers. Phylogenetic analysis using the small-subunit (SSU) ribosomal DNA (rDNA) data set revealed 22 different AMF sequence types (17 Glomus sequence types, 3 Paraglomus sequence types, 1 Scutellospora sequence type, and 1 Acaulospora sequence type). The highest AMF diversity was found in uninfected roots, followed by infected roots and galls. This study indicates that the galls produced in P. persica roots due to infection with M. incognita were colonized extensively by a community of AMF, belonging to the families Paraglomeraceae and Glomeraceae, that was different from the community detected in roots. Although the function of the AMF in the galls is still unknown, we hypothesize that they act as protection agents against opportunistic pathogens.     

Community structure of arbuscular mycorrhizal fungi in a primary successional volcanic desert on the southeast slope of Mount Fuji

Community structure of arbuscular mycorrhizal fungi (AMF), evaluated as spore samples and mycorrhizal roots of four herbaceous plant species, was investigated at different altitudes in a primary successional volcanic desert on Mount Fuji using molecular methods (fragment and sequence analysis of the large ribosomal subunit RNA gene). In total, 17 different AMF clades were identified, and most were members of the Glomaceae, Acaulosporaceae, and Gigasporaceae. The AMF community structures detected by spore sampling were inconsistent with those from plant roots. Of all AMF clades, six (35.3%) were detected only on the basis of spores, six (35.3%) only in roots, and five corresponded to both spores and roots (29.4%). Although an Acaulospora species was the most dominant among spores (67.1%), it accounted for only 6.8% in root samples. A species analysis of AMF communities at different altitudes demonstrated that AMF species diversity increased as altitude decreased and that the species enrichment at lower altitudes resulted from the addition of new species rather than species replacement. The inconsistencies in the species composition of spore communities with those in roots and the change in species diversity with altitude are discussed.     

Assessing the diversity of arbuscular mycorrhizal fungi in semiarid shrublands dominated by Artemisia tridentata ssp. wyomingensis

Variation in the abiotic environment and host plant preferences can affect the composition of arbuscular mycorrhizal (AMF) assemblages. This study analyzed the AMF taxa present in soil and seedlings of Artemisia tridentata ssp. wyomingensis collected from sagebrush steppe communities in southwestern Idaho, USA. Our aims were to determine the AMF diversity within and among these communities and the extent to which preferential AMF–plant associations develop during seedling establishment. Mycorrhizae were identified using molecular methods following DNA extraction from field and pot culture samples. The extracted DNA was amplified using Glomeromycota specific primers, and identification of AMF was based on phylogenetic analysis of sequences from the large subunit-D2 rDNA region. The phylogenetic analyses revealed seven phylotypes, two within the Claroideoglomeraceae and five within the Glomeraceae. Four phylotypes clustered with known species including Claroideoglomus claroideum, Rhizophagus irregularis, Glomus microaggregatum, and Funneliformis mosseae. The other three phylotypes were similar to several published sequences not included in the phylogenetic analysis, but all of these were from uncultured and unnamed glomeromycetes. Pairwise distance analysis revealed some phylotypes with high genetic variation. The most diverse was the phylotype that included R. irregularis, which contained sequences showing pairwise differences up to 12 %. Most of the diversity in AMF sequences occurred within sites. The smaller genetic differentiation detected among sites was correlated with differences in soil texture. In addition, multiplication in pot cultures led to differentiation of AMF communities. Comparison of sequences obtained from the soil with those from A. tridentata roots revealed no significant differences between the AMF present in these samples. Overall, the sites sampled were dominated by cosmopolitan AMF taxa, and young seedlings of A. tridentata ssp. wyomingensis were colonized in relation to the abundance of these taxa in the soil.     

Molecular evolution patterns reveal life history features of mycoplasma‐related endobacteria associated with arbuscular mycorrhizal fungi

The mycoplasma‐related endobacteria (MRE), representing a recently discovered lineage of Mollicutes, are widely distributed across arbuscular mycorrhizal fungi (AMF, Glomeromycota). AMF colonize roots of most terrestrial plants and improve plant mineral nutrient uptake in return for plant‐assimilated carbon. The role of MRE in the biology of their fungal hosts is unknown. To start characterizing this association, we assessed partitioning of MRE genetic diversity within AMF individuals and across the AMF phylogeographic range. We further used molecular evolution patterns to make inferences about MRE codivergence with AMF, their lifestyle and antiquity of the Glomeromycota–MRE association. While we did not detect differentiation between MRE derived from different continents, high levels of diversity were apparent in MRE populations within AMF host individuals. MRE exhibited significant codiversification with AMF over ecological time and the absence of codivergence over evolutionary time. Moreover, genetic recombination was evident in MRE. These patterns indicate that, while MRE transmission is predominantly vertical, their complex intrahost populations are likely generated by horizontal transmission and recombination. Based on predictions of evolutionary theory, we interpreted these observations as a suggestion that MRE may be antagonists of AMF. Finally, we detected a marginally significant signature of codivergence of MRE with Glomeromycota and the Endogone lineage of Mucoromycotina, implying that the symbiosis between MRE and fungi may predate the divergence between these two groups of fungi.     

A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase

Polymerase chain reaction (PCR) primers that flank a 688-bp segment within the glutamate dehydrogenase gene (gdh) of Streptococcus suis type 2 could amplify efficiently the DNA of all 306 (100%) clinical S. suis isolates tested (pigs, n=305; human, n=1) encompassing all serotypes obtained from diverse organs, and geographic origins. When DNA from other bacteria were used as templates for amplification, no product was detected indicating specificity of the primers. Multiplex PCR was developed using the gdh gene primer pair and primers that targeted the gene encoding S. suis capsular biosynthesis (cps). This strategy enabled the detection of strains belonging to serotypes 1/2, 1, 2, 7, and 9, respectively. Using the multiplex-PCR technique, 12 out of 14 (86%) isolates that were previously identified as non-typable S. suis (based on biochemical reactions and serology) gave positive PCR results of which four were positive for serotype 7, three for serotype 2, and five for S. suis strains that belong to other serotypes. Retest results of all 14 isolates by several veterinary laboratories were identical with PCR and confirmed that the two non-PCR reactive isolates belonged to strains of other streptococcal species. These results indicated that PCR improved species determination and can thus be used as a reliable species-specific molecular diagnostic reagent for the accurate identification of S. suis isolates and a serotype-specific method for the detection of strains of serotypes 1/2, 1, 2, 7, and 9, respectively. The PCR method therefore has potential clinical and epidemiological applications.     

Differences in the species composition of arbuscular mycorrhizal fungi in spore, root and soil communities in a grassland ecosystem

Most studies on the species composition of arbuscular mycorrhizal fungi (AMF) have solely analysed mycorrhizal roots or AM spores collected from soil samples. However, the spore production rate and proportions of AMF mycelium in roots and soils have all been shown to vary substantially in a taxon-specific manner. Therefore, in the study presented here we used a molecular approach to analyse the species composition of AMF in spores, intra-radical and extra-radical mycelium in an intensively farmed meadow in central Germany. By polymerase chain reaction and sequencing of the ITS region members of seven different families and species groups within Glomeromycota were identified. The data revealed remarkable differences in the composition of AMF taxa both between the spores and the mycelia, and between the two types of mycelia. Glomus group Ab was dominant in roots and spores, in accordance with previous research. However, members of this group were rarely detected as extra-radical mycelium, in which Paraglomeraceae were dominant, although we found no evidence for the presence of Paraglomeraceae in roots or spores, even when a specific primer set was used. These results may be interpreted as a further indication that AMF are not necessarily obligate symbionts of plants.     

Identification of endomycorrhizal fungi colonizing roots by fluorescent single-strand conformation polymorphism-polymerase chain reaction.

A method to identify arbuscular endomycorrhizal fungi based on the amplification of portions of the nuclear gene coding for the small subunit rRNA is presented. By coupling the sensitivity of the polymerase chain reaction and the specificity afforded by taxon-specific primers, a variety of samples can be analyzed, including small amounts of colonized roots. Family-specific primers as well as generic primers are described and can be used to amplify small subunit rRNA fragments from endomycorrhizal fungi by polymerase chain reaction. The amplified products are then subjected to single-strand conformation polymorphism analysis to detect sequence differences. Among the advantages of this approach is the possibility of directly identifying the fungi inside field-collected roots, without having to rely on the fortuitous presence of spores. This technique should have obvious applications in the study of arbuscular endomycorrhizal fungi populations and allow closer examination of their host specificity.     

Frankia genus-specific characterization by polymerase chain reaction.

The polymerase chain reaction (PCR) is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. In order to determine whether a given actinomycete isolated from an actinorhiza (nodule) belongs to the genus Frankia or is a contaminant, we have developed a test based on the PCR. Primers complementary to sequences of two DNA regions corresponding to the nif genes (nifH and nifD) and the rRNA genes (16S and 23S) were specifically chosen to differentially amplify DNAs from Frankia strains but not those from other microorganisms. A series of positive and negative controls were set up by using universal or selective primers resulting in a discriminant amplification, which could be detected after agarose gel electrophoresis. In the nif region, degenerate oligonucleotide primers were used to amplify a target common to all the nitrogen-fixing microorganisms tested, while another set of primers amplified a target with a high specificity for Frankia strains. In the rRNA gene region, universal and specific primers were characterized and tested with DNAs from a wide range of microorganisms. The efficiency of this rapid and sensitive PCR assay was tested with an isolate obtained from Alnus nepalensis nodules, confirming results obtained by nodulation tests.     

Frankia genus-specific characterization by polymerase chain reaction.

The polymerase chain reaction (PCR) is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. In order to determine whether a given actinomycete isolated from an actinorhiza (nodule) belongs to the genus Frankia or is a contaminant, we have developed a test based on the PCR. Primers complementary to sequences of two DNA regions corresponding to the nif genes (nifH and nifD) and the rRNA genes (16S and 23S) were specifically chosen to differentially amplify DNAs from Frankia strains but not those from other microorganisms. A series of positive and negative controls were set up by using universal or selective primers resulting in a discriminant amplification, which could be detected after agarose gel electrophoresis. In the nif region, degenerate oligonucleotide primers were used to amplify a target common to all the nitrogen-fixing microorganisms tested, while another set of primers amplified a target with a high specificity for Frankia strains. In the rRNA gene region, universal and specific primers were characterized and tested with DNAs from a wide range of microorganisms. The efficiency of this rapid and sensitive PCR assay was tested with an isolate obtained from Alnus nepalensis nodules, confirming results obtained by nodulation tests.     

ROLC strawberry plant adaptability, productivity, and tolerance to soil-borne disease and mycorrhizal interactions

The potential to improve strawberry cultivation was assessed regarding the use the rolC genes from Agrobacterium rhizogenes that can confer higher levels of free cytokinins. Strawberry (cv. Calypso) rolC lines were produced by genetic transformation of Agrobacterium tumefaciens. Yield and fruit quality of the control and transgenic lines were measured under open-field conditions. The effects of the transgenic rolC lines depended on gene copy number: rolC lines with one (Line A) or two gene (Line B) copies showed 30% greater yields than controls, due to 20% more fruit per plant and an increased fruit weight. Line A also differed in terms of the highest fruit quality, due to 10.5% increased soluble solids and 12.7% higher acidity. Moreover, cv. Calypso rolC lines A and B had increased tolerance to greenhouse infection by Phytophthora cactorum. Conversely, for all of these characters, Line F (five rolC copies) was not significantly different from the control line. The same lines were also used to examine their symbiosis with root arbuscular mycorrhizal fungi (AMF) using vital and non-vital staining of roots collected at different stages of plant growth. Control and rolC plants showed similar intensities of AMF infection according to plant phenology and/or physiology. Furthermore, possible horizontal gene transfer of the rolC gene was tested for the AMF spores by PCR, with all AMF samples negative using rolC primers. The use of the rolC gene should be considered for the improvements provided in productivity, fruit quality and disease resistance of cultivated strawberry that show no effects on soil microorganisms.     

Colonization of roots by arbuscular mycorrhizal fungi using different sources of inoculum

Arbuscular mycorrhizal fungi (AMF) form a number of different infective propagules that are used to form new mycorrhizal associations. These are spores, extraradical hyphae and infected roots. However, not all fungi are equally capable of colonizing roots with all of the above-mentioned propagules and there is conflicting evidence of major differences in colonization strategy between members of the Glomineae and Gigasporineae. In this study, we tested the abilities of eight fungal species from four different genera to colonize roots using three different types of inoculum. Glomus and Acaulospora isolates colonized from all inoculum types, whereas Gigaspora and Scutellospora isolates colonized mainly from spores and to a limited degree from root fragments. Extraradical hyphae were not suitable propagules for the species of Gigaspora and Scutellospora tested. This indicates that AMF have different colonization strategies and that this is largely differentiated at the suborder level. It is unclear why there is such a difference among the fungi in inoculum types. Future research should examine differences in the anatomy and physiology to discern a mechanism for such differences in life-history strategies.