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Choices on sampling, sequencing, and analyzing DNA influence the estimation of community composition of plant fungal symbionts

ABSTRACT: Abstract Plant root symbionts, namely mycorrhizal fungi, can be characterized using a variety of methods, but most of these rely on DNA. While Sanger sequencing still fulfills particular research objectives, next‐generation sequencing currently dominates the field, thus understanding how the two methods differ is important for identifying both opportunities and limitations to characterizing fungal communities. In addition to testing sequencing methods, we also examined how roots and soils may yield different fungal communities and how disturbance may affect those differences. We sequenced DNA from ectomycorrhizal fungi colonizing roots of Pinus banksiana and found that operational taxonomic unit richness was higher, and compositional variance lower, for Illumina MiSeq–sequenced communities compared to Sanger‐sequenced communities. We also found that fungal communities associated with roots were distinct in composition compared to those associated with soils and, moreover, that soil‐associated fungi were more clustered in composition than those of roots. Finally, we found community dissimilarity between roots and soils was insensitive to disturbance; however, rarefying read counts had a sizeable influence on trends in fungal richness. Although interest in mycorrhizal communities is typically focused on the abiotic and biotic filters sorting fungal species, our study shows that the choice of methods to sample, sequence, and analyze DNA can also influence the estimation of community composition.

SUBMITTER: Cale J

PROVIDER: S-EPMC8564097 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The emerging fungal pathogen, Batrachochytrium dendrobatidis (Bd), which can cause a fatal disease called chytridiomycosis, is implicated in the collapse of hundreds of host amphibian species. We describe chytridiomycosis dynamics in two co-occurring terrestrial salamander species, the Santa Lucia Mountains slender salamander, Batrachoseps luciae, and the arboreal salamander, Aneides lugubris. We (1) conduct a retrospective Bd-infection survey of specimens collected over the last century, (2) estimate present-day Bd infections in wild populations, (3) use generalized linear models (GLM) to identify biotic and abiotic correlates of infection risk, (4) investigate susceptibility of hosts exposed to Bd in laboratory trials, and (5) examine the ability of host skin bacteria to inhibit Bd in culture. Our historical survey of 2,866 specimens revealed that for most of the early 20th century (~1920-1969), Bd was not detected in either species. By the 1990s the proportion of infected specimens was 29 and 17% (B. luciae and A. lugubris, respectively), and in the 2010s it was 10 and 17%. This was similar to the number of infected samples from contemporary populations (2014-2015) at 10 and 18%. We found that both hosts experience signs of chytridiomycosis and suffered high Bd-caused mortality (88 and 71% for B. luciae and A. lugubris, respectively). Our GLM revealed that Bd-infection probability was positively correlated with intraspecific group size and proximity to heterospecifics but not to abiotic factors such as precipitation, minimum temperature, maximum temperature, mean temperature, and elevation, or to the size of the hosts. Finally, we found that both host species contain symbiotic skin-bacteria that inhibit growth of Bd in laboratory trials. Our results provide new evidence consistent with other studies showing a relatively recent Bd invasion of amphibian host populations in western North America and suggest that the spread of the pathogen may be enabled both through conspecific and heterospecific host interactions. Our results suggest that wildlife disease studies should assess host-pathogen dynamics that consider the interactions and effects of multiple hosts, as well as the historical context of pathogen invasion, establishment, and epizootic to enzootic transitions to better understand and predict disease dynamics.

Project description:BackgroundFungi associated with insects represent one potentially rich source for the discovery of novel metabolites. However, a comprehensive understanding of the fungal communities of Apis mellifera ligustica remains elusive.ResultsHere, we investigated the phylogenetic diversity and community composition of honeybee-associated fungi using combination of culture-dependent and culture-independent approaches. A total of forty-five fungi were isolated and purified from the Apis mellifera ligustica, royal jelly, and honeycomb, which belonged to four classes and eleven different genera. Furthermore, 28 bacterial 16S rRNA gene sequences were obtained by PCR from the fungal metagenome. High-throughput sequencing analyses revealed that the fungal communities were more diverse, a total of 62 fungal genera were detected in the honeybee gut by culture-independent method, whereas only 4 genera were isolated by culture-dependent method. Similarly, 247 fungal genera were detected in the honeycomb, whereas only 4 genera were isolated. In addition, we assessed the antibacterial and antioxidant activities of fungal isolates. Most fungal crude extracts obtained from the cultivation supernatant exhibited antioxidant activities. Only two fungal crude extracts displayed moderate activity against Escherichia coli and Staphylococcus aureus. Chemical analysis of Chaetomium subaffine MFFC22 led to the discovery of three known compounds, including cochliodinol (1), emodin (2), chrysophanol (3). Among them, cochliodinol (1) showed intense DPPH radical scavenging activity with the 50% inhibitory concentration (IC50) of 3.06 μg/mL, which was comparable to that of the positive ascorbic acid (IC50 = 2.25 μg/mL). Compound 2 displayed weak inhibitory activities against Micrococcus tetragenus and S. aureus.ConclusionsThis research provided a fundamental clue for the complex interactions among honeybees, fungi, bacterial symbionts, and the effects on the honeybee. Furthermore, the diversity of honeybee-associated fungi had great potential in finding the resource of new species and antioxidants.

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Choices on sampling, sequencing, and analyzing DNA influence the estimation of community composition of plant fungal symbionts

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