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.

• Klíčová slova
• Arbuscular mycorrhizal fungi, Isolate discrimination, Microsymbiont screening, Mitochondrial DNA, Molecular genetic quantification, Nuclear ribosomal DNA, PLFA, Real-time PCR,
• 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
• Názvy látek
• DNA fungální MeSH
• mitochondriální DNA MeSH

Monitoring populations of arbuscular mycorrhizal fungi (AMF) in roots is a pre-requisite for improving our understanding of AMF ecology and functioning of the symbiosis in natural conditions. Among other approaches, quantification of fungal DNA in plant tissues by quantitative real-time PCR is one of the advanced techniques with a great potential to process large numbers of samples and to deliver truly quantitative information. Its application potential would greatly increase if the samples could be preserved by drying, but little is currently known about the feasibility and reliability of fungal DNA quantification from dry plant material. We addressed this question by comparing quantification results based on dry root material to those obtained from deep-frozen roots of Medicago truncatula colonized with Rhizophagus sp. The fungal DNA was well conserved in the dry root samples with overall fungal DNA levels in the extracts comparable with those determined in extracts of frozen roots. There was, however, no correlation between the quantitative data sets obtained from the two types of material, and data from dry roots were more variable. Based on these results, we recommend dry material for qualitative screenings but advocate using frozen root materials if precise quantification of fungal DNA is required.

• MeSH
• DNA fungální genetika   izolace a purifikace   MeSH
• kořeny rostlin chemie   růst a vývoj   mikrobiologie   MeSH
• Medicago truncatula chemie   růst a vývoj   mikrobiologie   MeSH
• mykorhiza chemie   genetika   MeSH
• ochrana biologická MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH

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
• Názvy látek
• DNA fungální MeSH
• mitochondriální DNA MeSH