Workpackage 1

Fungal biodiversity from soil samples at Buffelskloof Nature reserve in South Africa

Dr. Hazal Kandemir

Westerdijk Fungal Biodiversity Institute, Evolutionary Phytopathology Group

The goal of this study is to provide an overview of the fungal diversity, including potentially novel species isolated from soil samples collected at the Buffelskloof Nature Reserve (BNR). Sampling was carried out during the field trip as a part of Work package 1 described as "Training of young researchers in taxonomic and phylogenetic studies of selected African and Asian fungi (M 1-24)" in the Mycobiomics project. Soil samples were collected from different locations in the BNR from the higher grassland to the lower riverbed, to compare the transition in fungal diversity along the altitude gradient. Fungal cultures were obtained via the dilution-to-extinction protocol. Preliminary identification of the isolates was done by amplification of the ITS and LSU regions, followed by secondary DNA barcodes depending on the fungal genus. The correlation between altitude, plant diversity and soil fungal diversity in the BNR will be evaluated, and DNA metabarcoding will be performed in the next step to obtain the overall fungal diversity compared to that obtained via cultivation practices. All cultures will be available in the CPC culture collection located at the Westerdijk Fungal Biodiversity Institute for further studies, such as exploring bioactive secondary metabolites from the isolates.

Participating institutes: Westerdijk Fungal Biodiversity Institute (WFBI) & Forestry and Agricultural Biotechnology Institute (FABI)

Workpackage 1

Training of young researchers in taxonomy, phylogeny and functional biodiversity of fungi

This work package includes academic exchange of young scientists who are jointly doing field work in Asia, Africa and Europe and evaluate the taxonomy of certain fungal groups, such as Xylariales and invertebrate-associated Hypocreales, as well as basidiomycetes. The fungi are being taken in pure culture and subjected to morphological, chemotaxonomic and molecular phylogenetic studies.

Examples

https://www.mdpi.com/2309-608X/9/4/429

https://mycokeys.pensoft.net/article/98125/

Workpackage 2

Studies of invertebrate- and plant-associated fungi for bioactive secondary metabolites

This work package encompasses intensive studies on ecologically and economically important taxonomic groups of fungi, with which the partners of the Mycobiomics consortium are fairly familiar. Aside from the taxa mentioned in WP1 (Xylariales and invertebrate-associated Hypocreales), we are also addressing hitherto neglected fungal symbionts of wood-inhabiting insects. The strains of these fungi are being studied for secondary metabolites. The identified molecules are being tested for biological activities in the work packages 4-5, but notably they are also being investigated for their potential utility as chemotaxonomic markers.

XExamples: https://link.springer.com/article/10.1007/s11557-023-01915-3

Figure: (Click on the image to open it as a PDF document in a new window)

Workpackage 3

Comparison of Fusarium diversity, metabolites and genomes from maize, and search for secondary metabolites produced by its natural antagonists

In this work package, the speices of the important genus Fusarium and its allies are being studied for taxonomy, phylogeny and mycotoxin production. Strains from Europe, Africa and Thailand will be checked for secondary metabolites an the biosynthetic gene clusters encoding for their production are being identified. Another task is the search for new active compounds from the natural antagonists of Fusarium spp.

XExamples: https://pubmed.ncbi.nlm.nih.gov/34466168/

Figure: (Click on the image to open it as a PDF document in a new window)

Workpackage 4

Secondary metabolite driven communication and antagonism elicited by fungal co-cultures with other microbes

Objective: Search for novel natural defence compounds against fungal pathogens in dual culture.

Based on the newly isolated strains from WP1-3, a high throughput dual culturing program with different microbes and fungi is being carried out at BOKU. Fermentations of strains that show production of active compounds as a defense reaction in dual culture will be scaled up to isolate and identify the active principles, which will also be tested in a series of other bioassays, once they have been purified.

Workpackage 5

Intensive biological characterization of the identified secondary metabolites including studies on their mode of action

Selected fungal strains are being subjected to fermentation in larger scale and their cultures are extracted to isolate the secondary metabolites. These compounds are being tested for selectivity e.g., against bacteria, fungi and mammalian cells and their chemical structures are elucidated.

Examples

https://www.mdpi.com/2079-6382/11/8/1072

https://pubs.acs.org/doi/10.1021/acs.jnatprod.2c01015

Workpackage 6

Identification of secondary metabolite gene clusters and functional analysis of secondary metabolite production

The genomes of selected strains are being sequenced and aside from phylogenomic studies, the biosynthetic gene clusters (BGC) encoding for secondary metabolite production are being identified. Selected BGC are being subjected to heterologous expression and other functional analyses. The first genomes have already been sequenced, and some papers have already resulted from the joint project work on genome analyses.

Examples

https://imafungus.biomedcentral.com/articles/10.1186/s43008-022-00103-4

https://www.frontiersin.org/articles/10.3389/fmicb.2023.1108975/full

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403587/

(This work was done before the Mycobiomics project started but the content is related to it.)

Workpackage 7

Isolation and analysis of endophytic Trichoderma strains and plant interactions

Endophytic and saprotrophic Trichoderma strains are being tested for their efficiency in plant protection; at a later stage it is planned to evaluate relevant secondary metabolites and marker genes and correlate those with plant protection abilities. The first experiments are presently ongoing.

Workpackage 8

Scale up of Fermentation/Production

Task 8.1 Optimisation of production/downstream processing for selected strains

Out of the most promising strains resulting from WP 1-7, five to ten will be selected for large scale fermentation, using the pilot plant bioreactors available at HZI and scale-up processes will be developed up to 350 litre scale. Aside from the fermentation, the downstream processing involving different methods for extraction and chromatography will also be optimized. This work is planned in the third year of the project.