Crop inoculation with Glomus cubense isolate (INCAM-4, DAOM-241198) promotes yield in banana, cassava, forages, and others. Yield improvements range from 20 to 80% depending on crops, nutrient supply, and edaphoclimatic conditions. However, it is difficult to connect yield effects with G. cubense abundance in roots due to the lack of an adequate methodology to trace this taxon in the field. It is necessary to establish an accurate evaluation framework of its contribution to root colonization separated from native arbuscular mycorrhizal fungi (AMF). A taxon-discriminating primer set was designed based on the ITS nrDNA marker and two molecular approaches were optimized and validated (endpoint PCR and quantitative real-time PCR) to trace and quantify the G. cubense isolate in root and soil samples under greenhouse and environmental conditions. The detection limit and specificity assays were performed by both approaches. Different 18 AMF taxa were used for endpoint PCR specificity assay, showing that primers specifically amplified the INCAM-4 isolate yielding a 370 bp-PCR product. In the greenhouse, Urochloa brizantha plants inoculated with three isolates (Rhizophagus irregularis, R. clarus, and G. cubense) and environmental root and soil samples were successfully traced and quantified by qPCR. The AMF root colonization reached 41-70% and the spore number 4-128 per g of soil. This study demonstrates for the first time the feasibility to trace and quantify the G. cubense isolate using a taxon-discriminating ITS marker in roots and soils. The validated approaches reveal their potential to be used for the quality control of other mycorrhizal inoculants and their relative quantification in agroecosystems.
Ericoid mycorrhiza is arguably the least investigated mycorrhizal type, particularly when related to the number of potential hosts and the ecosystems they inhabit. Little is known about the global distribution of ericoid mycorrhizal (ErM) fungi, and this holds true even for the prominent ErM mycobiont Rhizoscyphus ericae. Earlier studies suggested R. ericae might be low in abundance or absent in the roots of Southern Hemisphere's Ericaceae, and our previous investigations in two Argentine Patagonian forests supported this view. Here, we revisited the formerly investigated area, albeit at a higher altitude, and screened fungi inhabiting hair roots of Gaultheria caespitosa and Gaultheria pumila at a treeless alpine site using the same methods as previously. We obtained 234 isolates, most of them belonging to Ascomycota. In contrast to previous findings, however, among 37 detected operational taxonomic units (OTUs), OTU 1 (=R. ericae s. str.) comprised the highest number of isolates (87, ∼37 %). Most of the OTUs and isolates belonged to the Helotiales, and 82.5 % of isolates belonged to OTUs shared between both Gaultheria species. At the alpine site, ericoid mycorrhizal fungi dominated, followed by dark septate endophytes and aquatic hyphomycetes probably acting as root endophytes. Our results suggest that the distribution of R. ericae is influenced, among others, by factors related to altitude such as soil type and presence/absence and type of the neighboring vegetation. Our study is the first report on R. ericae colonizing Ericaceae roots in the Southern Hemisphere and extends the known range of this prominent ErM species to NW Patagonia.
- MeSH
- Ericaceae mikrobiologie MeSH
- fylogeneze MeSH
- Glomeromycota klasifikace genetika izolace a purifikace MeSH
- kořeny rostlin mikrobiologie MeSH
- mykorhiza fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Argentina MeSH
Root colonization by arbuscular mycorrhizal fungi (AMF) can be quantified by different approaches. We compared two approaches that enable discrimination of specific AMF taxa and are therefore emerging as alternative to most commonly performed microscopic quantification of AMF in roots: quantitative real-time PCR (qPCR) using markers in nuclear ribosomal DNA (nrDNA) and mitochondrial ribosomal DNA (mtDNA). In a greenhouse experiment, Medicago truncatula was inoculated with four isolates belonging to different AMF species (Rhizophagus irregularis, Claroideoglomus claroideum, Gigaspora margarita and Funneliformis mosseae). The AMF were quantified in the root samples by qPCR targeted to both markers, microscopy and contents of AMF-specific phospholipid fatty acids (PLFA). Copy numbers of nrDNA and mtDNA were closely related within all isolates; however, the slopes and intercepts of the linear relationships significantly differed among the isolates. Across all isolates, a large proportion of variance in nrDNA copy numbers was explained by root colonization intensity or contents of AMF-specific PLFA, while variance in mtDNA copy numbers was mainly explained by differences among AMF isolates. We propose that the encountered inter-isolate differences in the ratios of mtDNA and nrDNA copy numbers reflect different physiological states of the isolates. Our results suggest that nrDNA is a more suitable marker region than mtDNA for the quantification of multiple AMF taxa as its copy numbers are better related to fungal biomass across taxa than are copy numbers of mtDNA.
- MeSH
- buněčné jádro genetika MeSH
- DNA fungální genetika MeSH
- Glomeromycota genetika MeSH
- kořeny rostlin mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce * MeSH
- Medicago truncatula mikrobiologie MeSH
- mitochondriální DNA genetika MeSH
- mykorhiza genetika MeSH
- Publikační typ
- časopisecké články MeSH
The Archaeorhizomycetes are recently discovered fungi with poorly resolved ecology. Even their abundance in soil fungal communities is currently disputed. Here we applied a PCR-independent, RNA-based metatranscriptomic approach to determine their abundance among fungi in eleven different soils across Europe. Using small subunit (SSU) ribosomal RNA transcripts as marker, we detected Archaeorhizomycetes in 17 out of 28 soil metatranscriptomes. They had average relative SSU rRNA abundance of 2.0% with a maximum of 9.4% among fungal SSU rRNAs. Network analysis revealed that they co-occur with arbuscular mycorrhizal Glomerales, which is in line with their previously suggested association with plant roots. Moreover, Archaeorhizomycetes ranked among the potential keystone taxa. This metatranscriptomic survey exemplifies the usage of non-targeted molecular approaches for the study of soil fungi. It provides PCR- and DNA-independent evidence for the low abundance of Archaeorhizomycetes in soil fungal communities, although they might be non-negligible players despite their low abundance.
Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.
- MeSH
- DNA fungální chemie genetika MeSH
- Glomeromycota genetika růst a vývoj MeSH
- kořeny rostlin mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- Medicago sativa mikrobiologie MeSH
- mikrobiální interakce MeSH
- mitochondriální DNA genetika MeSH
- molekulární sekvence - údaje MeSH
- počet mikrobiálních kolonií metody MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
During the last decade, the application of arbuscular mycorrhizal fungi (AMF) as bioenhancers has increased significantly. However, until now, it has been difficult to verify the inoculation success in terms of fungal symbiont establishment in roots of inoculated plants because specific fungal strains could not be detected within colonized roots. Using mitochondrial large subunit ribosomal DNA, we show that Rhizophagus irregularis (formerly known as Glomus intraradices) isolate BEG140 consists of two different haplotypes. We developed nested PCR assays to specifically trace each of the two haplotypes in the roots of Phalaris arundinacea from a field experiment in a spoil bank of a former coal mine, where BEG140 was used as inoculant. We revealed that despite the relatively high diversity of native R. irregularis strains, R. irregularis BEG140 survived and proliferated successfully in the field experiment and was found significantly more often in the inoculated than control plots. This work is the first one to show tracing of an inoculated AMF isolate in the roots of target plants and to verify its survival and propagation in the field. These results will have implications for basic research on the ecology of AMF at the intraspecific level as well as for commercial users of mycorrhizal inoculation.
- MeSH
- DNA fungální genetika MeSH
- fylogeneze MeSH
- genetické markery MeSH
- Glomeromycota genetika izolace a purifikace fyziologie MeSH
- haplotypy MeSH
- kořeny rostlin mikrobiologie MeSH
- mitochondriální DNA genetika MeSH
- mitochondrie genetika MeSH
- molekulární sekvence - údaje MeSH
- mykorhiza genetika izolace a purifikace fyziologie MeSH
- Phalaris mikrobiologie fyziologie MeSH
- polymerázová řetězová reakce metody MeSH
- půdní mikrobiologie MeSH
- ribozomální DNA genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- sekvenční seřazení MeSH
- symbióza MeSH
- těžba uhlí MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
