Effects of one dark septate endophytic fungal and two Helotiales strains on the growth of Salix planifolia cuttings on iron ore waste rock

Plants maintain beneficial mutualistic relationships with the mycobiont communities found in their rhizosphere, leading to an increase in plant productivity and health. In nutrient-depleted substrates like mine tailings, mycobiont inoculation is often recommended to help restore a successful plant cover. Our 15-week greenhouse experiment aimed to assess the individual effects of a dark septate endophyte (Phialocephala fortinii #4; KX611529) and two Helotiales strains (Rhizoscyphus ericae #22; EU221877 and Meliniomyces sp #1; KT275679) on the growth of Salix planifolia cuttings on sterilized and unsterilized waste rock. Rhizoscyphus ericae increased cuttings shoot biomass on sterilized waste rock while Meliniomyces sp had a positive effect for cuttings grown on unsterilized waste rock. However, P. fortinii strain had no effect on the survival rate, shoot production, and biomass production of S. planifolia cuttings. This study demonstrates that controlled inoculation with ecologically well-adapted mycobionts could promote plant establishment and productivity on abandoned waste rock and be an efficient and integrated biotechnological approach for ecological restoration of canadian mining boreal ecosystems.

An Efficient Genetic Manipulation Protocol for Dark Septate Endophyte Falciphora Oryzae

Falciphora oryzae is a dark septate endophyte (DSE) isolated from wild rice roots (Oryza sativa L.). It was classified as a non-clavicitaceous endophyte. The fungus colonizes rice roots, showing a significant increase in agronomic parameters with plant biomass, rice blast resistance, yield, and quality. The construction of the genetic manipulation system is critical to study the relationship between F. oryzae and O. sativa. In the present study, the protoplast preparation and transformation system of F. oryzae was investigated. The key parameters affecting the efficiency of protoplast production, such as osmotic pressure stabilizer, enzyme digestion conditions, and fungal age, were studied. The results showed that F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9mol L-1 KCl solution and 10 mg mL−1 glucanase at 30℃ with shaking 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar (RgA) medium containing 1M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired GFP-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots.The genetic manipulation system of endophytic fungus facilitates the further exploration the interaction between the endophytic fungus and their hosts.

Bioerosion and fungal colonization of the invasive foraminiferan <i>Amphistegina lobifera</i> in a Mediterranean seagrass meadow

Foraminiferans are diverse micro- to macroscopic protists abundant especially in (sub)tropical seas, often forming characteristic benthic communities known as “living sands”. Numerous species have migrated through the Suez Canal to the Mediterranean and one of them, i.e., Amphistegina lobifera, turned invasive, gradually outcompeting the indigenous species. At some places, A. lobifera creates thick seabed sediments, thus becoming an important environmental engineer. However, little is known about the turnover of its shells in the invaded ecosystems. Using vital staining, stereomicroscopy, scanning electron microscopy, and cultivation and DNA fingerprinting, I investigated the vital status, destruction/decomposition and mycobiota of A. lobifera in the rhizosphere of the dominant Mediterranean seagrass Posidonia oceanica in an underwater Maltese meadow (average 284 shells g−1, representing 28.5 % of dry substrate weight), in comparison with epiphytic specimens and P. oceanica roots. While 78 % of the epiphytes were alive, nearly all substrate specimens were dead. On average, 80 % of the epiphytes were intact compared to 21 % of the substrate specimens. Abiotic dissolution and mechanical damage played only a minor role, but some bioerosion was detected in 18 % and >70 % of the epiphytic and substrate specimens, respectively. Few bioerosion traces could be attributed to fungi, and the majority probably belonged to photoautotrophs. The seagrass roots displayed fungal colonization typical for this species and yielded 81 identified isolates, while the surface-sterilized substrate specimens surprisingly yielded no cultivable fungi compared to 16 other identified isolates obtained from the epiphytes. While the epiphytes' mycobiota was dominated by ascomycetous generalists also known from terrestrial ecosystems (alongside with, for example, a relative of the “rock-eating” extremophiles), the roots were dominated by the seagrass-specific dark septate endophyte Posidoniomyces atricolor and additionally contained a previously unreported lulworthioid mycobiont. In conclusion, at the investigated locality, dead A. lobifera shells seem to be regularly bioeroded by endolithic non-fungal organisms, which may counterbalance their accumulation in the seabed substrate.

Near-chromosome-level genome assembly of the dark septate endophyte Laburnicola rhizohalophila: a model for investigating root-fungus symbiosis

The novel DSE Laburnicola rhizohalophila (Pleosporales, Ascomycota) is frequently found in the halophytic seepweed (Suaeda salsa). In this paper, we report a near-chromosome-level hybrid assembly of this fungus using a combination of short-read Illumina data to polish assemblies generated from long-read Nanopore data. The reference genome for L. rhizohalophila was assembled into 26 scaffolds with a total length of 64.0 Mb and a N50 length of 3.15 Mb. Of them, 17 scaffolds approached the length of intact chromosomes, and 5 had telomeres at one end only. A total of 10,891 gene models were predicted. Intriguingly, 27.5 Mb of repeat sequences that accounted for 42.97% of the genome was identified, and long terminal repeat retrotransposons were the most frequent known transposable elements (TEs), indicating that TE proliferation contributes to its increased genome size. BUSCO analyses using the Fungi_odb10 dataset showed that 95.0% of genes were complete. In addition, 292 carbohydrate active enzymes, 33 secondary metabolite clusters, and 84 putative effectors were identified in silico. The resulting high-quality assembly and genome features are not only an important resource for further research on understanding the mechanism of root-fungi symbiotic interactions, but will also contribute to comparative analyses of genome biology and evolution within Pleosporalean species.

Melatonin confers heavy metal-induced tolerance by alleviating oxidative stress and reducing the heavy metal accumulation in Exophiala pisciphila, a dark septate endophyte (DSE)

Background Melatonin (MT), ubiquitous in almost all organisms, functions as a free radical scavenger. Despite several reports on its role as an antioxidant in animals, plants, and some microorganisms, extensive studies in filamentous fungi are limited. Based upon the role of melatonin as an antioxidant, we investigated its role in heavy metal-induced stress tolerance in Exophiala pisciphila, a dark septate endophyte (DSE), by studying the underlying mechanisms in alleviating oxidative stress and reducing heavy metal accumulation. Results A significant decrease in malondialdehyde (MDA) and oxygen free radical (OFR) in E. pisciphila was recorded under Cd, Zn, and Pb stresses as compared to the control. Pretreatment of E. pisciphila with 200.0 μM exogenous melatonin significantly increased the activity of superoxide dismutase (SOD) under Zn and Pb stresses. Pretreatment with 200.0 μM melatonin also lowered Cd, Zn, and Pb concentrations significantly. Melatonin production was enhanced by Cd, Cu, and Zn after 2 d, and melatonin biosynthetic enzyme genes, E. pisciphila tryptophan decarboxylase (EpTDC1) and serotonin N-acetyltransferase (EpSNAT1), were transcriptionally upregulated. The overexpression of EpTDC1 and N-acetylserotonin O-methyltransferase (EpASMT1) in Escherichia coli and Arabidopsis thaliana enhanced its heavy metal-induced stress tolerance. The overexpression of EpTDC1 and EpASMT1 reduced the Cd accumulation in the whole A. thaliana plants, especially in the roots. Conclusions Melatonin conferred heavy metal-induced stress tolerance by alleviating oxidative stress, activating antioxidant enzyme SOD, and reducing heavy metal accumulation in E. pisciphila. Melatonin biosynthetic enzyme genes of E. pisciphila also played key roles in limiting excessive heavy metal accumulation in A. thaliana. These findings can be extended to understand the role of melatonin in other DSEs associated with economically important plants and help develop new strategies in sustainable agriculture practice where plants can grow in soils contaminated with heavy metals.