Orthogonal protocols for DNA extraction from filamentous fungi

Conlon, Benjamin H.; Schmidt, Suzanne; Poulsen, Michael; Shik, Jonathan Z.

doi:10.1016/j.xpro.2022.101126

Cited by 14 publications

(4 citation statements)

References 3 publications

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“…A second experiment repeated this approach but added antibiotics (ampicillin, chloramphenicol and streptomycin) (for concentrations, see: [ 22 ]) to each treatment ( n = 15 per treatment) and was performed over 52 days. We confirmed that bacteria were excluded from antibiotic-treated plates by collecting fungal mycelia from the antibiotic-treated and control plates and extracting DNA using a Chelex ® (Sigma-Aldrich, USA) protocol [ 23 , 24 ]. DNA for positive controls was extracted, using the same method, from pure colonies of bacteria: Streptomyces sp.…”

Section: Methodsmentioning

confidence: 99%

A fungal symbiont converts provisioned cellulose into edible yield for its leafcutter ant farmers

et al. 2022

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While ants are dominant consumers in terrestrial habitats, only the leafcutters practice herbivory. Leafcutters do this by provisioning a fungal cultivar ( Leucoagaricus gongylophorus ) with freshly cut plant fragments and harnessing its metabolic machinery to convert plant mulch into edible fungal tissue (hyphae and swollen hyphal cells called gongylidia). The cultivar is known to degrade cellulose, but whether it assimilates this ubiquitous but recalcitrant molecule into its nutritional reward structures is unknown. We use in vitro experiments with isotopically labelled cellulose to show that fungal cultures from an Atta colombica leafcutter colony convert cellulose-derived carbon into gongylidia, even when potential bacterial symbionts are excluded. A laboratory feeding experiment showed that cellulose assimilation also occurs in vivo in A. colombica colonies. Analyses of publicly available transcriptomic data further identified a complete, constitutively expressed, cellulose-degradation pathway in the fungal cultivar. Confirming leafcutters use cellulose as a food source sheds light on the eco-evolutionary success of these important herbivores.

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Section: Methodsmentioning

confidence: 99%

A fungal symbiont converts provisioned cellulose into edible yield for its leafcutter ant farmers

et al. 2022

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“…We extracted DNA from 17 Termitomyces isolates using a CTAB extraction optimised for high yield and fragment length, with an initial fast-freeze step to increase DNA yield 21 . Wholegenome sequencing was performed using a combination of 100bp/150bp paired-end shotgun (BGISEQ/DNBSEQ) and long-read (PacBio Sequel) sequencing by BGI.…”

Section: Genome Sequencing Assembly and Annotationmentioning

confidence: 99%

“…New isolates were verified as Termitomyces by barcoding of the Internal Transcribed Spacer (ITS) region 20 . DNA was extracted using the Chelex protocol as described by Conlon (2022) 21 .…”

Section: Strain Genotypingmentioning

confidence: 99%

Comparative genomics unravels a rich set of biosynthetic gene clusters with distinct evolutionary trajectories across fungal species farmed by termites

Schmidt,

Murphy,

Vizueta

et al. 2024

Preprint

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The use of compounds produced by hosts or symbionts for defence against antagonists has been identified in many organisms, including in fungus-farming termites (Macrotermitinae). The obligate mutualistic fungus Termitomyces plays a central role in the symbiosis through plant biomass decomposition and as the main food source for these termites. Several specialised (secondary) metabolites have been isolated from different Termitomyces species, suggesting that they may also aid in antimicrobial defence. Yet, we have a fragmented understanding of Termitomyces’ natural product repertoire. To determine the biochemical potential encoded by diverse Termitomyces species, we comparatively analysed 22 published and 17 newly generated genomes, spanning 21 of 52 described Termitomyces species and five of the 11 termite host genera. After extensive assembly and annotation optimisation, we employed fungiSMASH to detect 754 biosynthetic gene clusters (BGCs) coding for specialised metabolites. BiG-SCAPE analysis and manual curation allowed us to assign 660 of these BGCs to 61 distinct biosynthetic gene cluster families (GCFs), spanning five compound classes. Seven GCFs were shared by all 21 Termitomyces species, 21 GCFs were present in all genomes of several subsets of species, while the remaining 33 GCFs were inconsistently distributed across species. The 25 most abundant GCFs were subjected to codon-based evolutionary constraint analyses to evaluate their evolutionary histories and revealed two GCFs with consistent positive selection in the same gene across the phylogeny and seventeen genes with Termitomyces species-specific episodic positive selection. These patterns of selection indicate that millions of years of termite-fungus symbiosis have led to distinct evolutionary trajectories of biosynthetic gene clusters, ample putative chemical novelties, and uncover a vast non-random and largely unknown chemical potential of Termitomyces.

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“…We verified species identities of the fungal strains by sequencing the Internal Transcribed Spacer (ITS) region of the nuclear ribosomal DNA (Schoch et al, 2012). For DNA isolation, we used a Chelex protocol as described in Conlon et al (2022). We used either the basidiomycete-specific primers ITS1F and ITS4B (Gardes and Bruns, 1993) or the ascomycete-specific primers ITS5F and ITS4B (White et al, 1990).…”

Section: Strain Genotypingmentioning

confidence: 99%

Make the environment protect you from disease: elevated CO2 inhibits antagonists of the fungus-farming termite symbiosis

et al. 2023

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Fungus-farming termite colonies maintain monoculture fungus combs in underground chambers without apparent problems with diseases. Multiple lines of defense contribute to the suppression or removal of antagonists of the symbiosis, but the role of the termite-manipulated environment within mounds has yet to be tested. Specifically, termite mounds have extremely high levels of CO2 compared to atmospheric levels. We tested the effect of 5% CO2 on the growth of fungal crops from Macrotermes bellicosus colonies, generalist fungi that could challenge the symbiosis, as well as a specialist stowaway fungus, Pseudoxylaria. For sporulating fungi, we also quantified the effects on conidia production. We found that elevated CO2 significantly reduces mycelial growth and conidia production of the generalist fungi Aspergillus sp., Beauveria bassiana, and Metarhizium brunneum, whereas it overall had a net positive effect on the growth of the fungal crop Termitomyces and Pseudoxylaria; albeit, with variation between fungal strains within genera. Our findings point to elevated CO2 being of adaptive significance to the fungus-farming termite symbiosis as an additional layer of defense that helps keep termite fungus gardens free from fungal infections. The mound-building activities that make termites ecosystem engineers may thus also generate environmental conditions that impact the fate of fungi inhabiting the extended phenotypes that massive termite mounds represent.

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Orthogonal protocols for DNA extraction from filamentous fungi

Cited by 14 publications

References 3 publications

A fungal symbiont converts provisioned cellulose into edible yield for its leafcutter ant farmers

A fungal symbiont converts provisioned cellulose into edible yield for its leafcutter ant farmers

Comparative genomics unravels a rich set of biosynthetic gene clusters with distinct evolutionary trajectories across fungal species farmed by termites

Make the environment protect you from disease: elevated CO2 inhibits antagonists of the fungus-farming termite symbiosis

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