Impact of dark septate endophytes on salt stress alleviation of tomato plants.

Fungal endophytes can improve plant tolerance to abiotic stress conditions. Dark septate endophytes (DSEs) belong to phylogenetically non-related groups of root colonizing fungi among the Ascomycota with high melanin-producing activities. They can be isolated from roots of more than 600 plant species in diverse ecosystems. Still the knowledge about their interaction with host plants and their contribution to stress alleviation is limited. The current work aimed to test the abilities of three DSEs (Periconia macrospinosa, Cadophora sp., Leptodontidium sp.) to alleviate moderate and high salt stress in tomato plants. By including an albino mutant, the role of melanin for the interaction with plants and salt stress alleviation could also be tested. P. macrospinosa and Cadophora sp. improved shoot and root growth 6 weeks after inoculation under moderate and high salt stress conditions. No matter how much salt stress was applied, macroelement (P, N, and C) contents were unaffected by DSE inoculation. The four tested DSE strains successfully colonized the roots of tomato, but the colonization level was clearly reduced in the albino mutant of Leptodontidium sp. Any difference in the effects on plant growth between the Leptodontidium sp. wild type strain and the albino mutant could, however, not be observed. These results show that particular DSEs are able to increase salt tolerance as they promote plant growth specifically under stress condition. Increased plant biomasses combined with stable nutrient contents resulted in higher P uptake in shoots of inoculated plants at moderate and high salt conditions and higher N uptake in the absence of salt stress in all inoculated plants, in P. macrospinosa-inoculated plants at moderate salt condition and in all inoculated plants except the albino mutants at high salt condition. In summary, melanin in DSEs seems to be important for the colonization process, but does not influence growth, nutrient uptake or salt tolerance of plants.

Salt Stress Tolerance of Dark Septate Endophytes Is Independent of Melanin Accumulation.

Dark septate endophytes (DSEs) represent a diverse group of root-endophytic fungi that have been isolated from plant roots in many different natural and anthropogenic ecosystems. Melanin is widespread in eukaryotic organisms and possesses various functions such as protecting human skin from UV radiation, affecting the virulence of pathogens, and playing a role in development and physiology of insects. Melanin is a distinctive feature of the cell walls of DSEs and has been thought to protect these fungi from abiotic stress. Melanin in DSEs is assumed to be synthesized via the 1,8-dihydroxynaphthalene (DHN) pathway. Its function in alleviation of salt stress is not yet known. The aims of this study were: (i) investigating the growth responses of three DSEs (Periconia macrospinosa, Cadophora sp., and Leptodontidium sp.) to salt stress, (ii) analyzing melanin production under salt stress and, (iii) testing the role of melanin in salt stress tolerance of DSEs. The study shows that the three DSE species can tolerate high salt concentrations. Melanin content increased in the hyphae of all DSEs at 100 mM salt, but decreased at 500 mM. This was not reflected in the RNA accumulation of the gene encoding scytalone dehydratase which is involved in melanin biosynthesis. The application of tricyclazole, a DHN-melanin biosynthesis inhibitor, did not affect either salt stress tolerance or the accumulation of sodium in the hyphae. In addition, melanin biosynthesis mutants of Leptodontidium sp. did not show decreased growth performance compared to the wild-type, especially not at high salt concentrations. This indicates that DSEs can live under salt stress and withstand these conditions regardless of melanin accumulation.

Unravelling the Role of Melanin in Cd and Zn Tolerance and Accumulation of Three Dark Septate Endophytic Species.

Dark septate endophytes (DSEs) are often trace element (TE)-tolerant fungi and are abundant in TE-polluted environments. The production of melanin, a black polymer found in cell walls, was hypothesized by several authors to play a role in the TE tolerance of DSEs. To test this hypothesis, we established a series of experiments using albino strains and melanin inhibitors and examined the responses to Cd and Zn. Six DSEs belonging to genera Cadophora sp., Leptodontidium sp. and Phialophora mustea, were evaluated. The strains mainly produced 1,8-dihydroxynaphthalene (DHN) melanin whereas 3,4-dihydroxyphenylalanin melanin was also synthetized. Cd and Zn decreased melanin synthesis in most of the strains. A reduction in melanin concentration in hyphae through the use of tricyclazole, an inhibitor of DHN-melanin synthesis, did not reduce the tolerance of the strains to Cd and Zn. Similarly, albino mutants of Leptodontidium sp. were not more sensitive to Cd and Zn than the WT strain. Moreover, tricyclazole-treated colonies accumulated less Cd but more Zn compared to untreated colonies. The Cd and Zn contents of Leptodontidium albino strains were variable and similar to that of the WT. The results suggest that melanin production is not an important functional trait that contributes to Cd and Zn tolerance, but might contribute to Cd accumulation.

Interactions between dark septate endophytes, ectomycorrhizal fungi and root pathogens in vitro.

Dark septate endophytes (DSEs) are widely distributed worldwide and can promote plant growth. Therefore, they are considered potentially important plant allies, especially in stressful environments. Previous studies have reported that DSEs cohabit roots with other microorganisms such as ectomycorrhizal (ECM), endophytic and pathogenic fungi/oomycetes. However, interactions between different DSE species have not yet been reported, and studies on the interactions between DSEs and other fungi are scarce. Using a simple and reproducible pairwise growth assay in vitro, we studied the synergistic/antagonistic interactions between eight DSEs, two ECM fungi and three root pathogens. Most of the DSE/DSE outcomes were neutral. Interestingly, we identified several DSE strains acting in synergy with other strains, as well as strains that could potentially act as biocontrol agents. Notably, three metal-tolerant DSE strains, namely, Cadophora sp., Leptodontidium sp. and Phialophora mustea, could decrease the growth of the root phytopathogens Pythium intermedium, Phytophthora citricola and Heterobasidion annosum. The present data are discussed in the general context of the use of fungal consortia as inocula in the tree-based phytomanagement of marginal lands.

Cadophora margaritata sp. nov. and other fungi associated with the longhorn beetles Anoplophora glabripennis and Saperda carcharias in Finland.

Symbiosis with microbes is crucial for survival and development of wood-inhabiting longhorn beetles (Coleoptera: Cerambycidae). Thus, knowledge of the endemic fungal associates of insects would facilitate risk assessment in cases where a new invasive pest occupies the same ecological niche. However, the diversity of fungi associated with insects remains poorly understood. The aim of this study was to investigate fungi associated with the native large poplar longhorn beetle (Saperda carcharias) and the recently introduced Asian longhorn beetle (Anoplophora glabripennis) infesting hardwood trees in Finland. We studied the cultivable fungal associates obtained from Populus tremula colonised by S. carcharias, and Betula pendula and Salix caprea infested by A. glabripennis, and compared these to the samples collected from intact wood material. This study detected a number of plant pathogenic and saprotrophic fungi, and species with known potential for enzymatic degradation of wood components. Phylogenetic analyses of the most commonly encountered fungi isolated from the longhorn beetles revealed an association with fungi residing in the Cadophora-Mollisia species complex. A commonly encountered fungus was Cadophora spadicis, a recently described fungus associated with wood-decay. In addition, a novel species of Cadophora, for which the name Cadophora margaritata sp. nov. is provided, was isolated from the colonised wood.

Co-inoculation of Lolium perenne with Funneliformis mosseae and the dark septate endophyte Cadophora sp. in a trace element-polluted soil.

The presence of dark septate endophytes (DSEs) or arbuscular mycorrhizal fungi (AMF) in plant roots and their effects on plant fitness have been extensively described. However, little is known about their interactions when they are simultaneously colonizing a plant root, especially in trace element (TE)-polluted soils. We therefore investigated the effects of Cadophora sp. and Funneliformis mosseae on ryegrass (Lolium perenne) growth and element uptake in a Cd/Zn/Pb-polluted soil. The experiment included four treatments, i.e., inoculation with Cadophora sp., inoculation with F. mosseae, co-inoculation with Cadophora sp. and F. mosseae, and no inoculation. Ryegrass biomass and shoot Na, P, K, and Mg concentrations significantly increased following AMF inoculation as compared to non-inoculated controls. Similarly, DSE inoculation increased shoot Na concentration, whereas dual inoculation significantly decreased shoot Cd concentration. Moreover, oxidative stress determined by ryegrass leaf malondialdehyde concentration was alleviated both in the AMF and dual inoculation treatments. We used quantitative PCR and microscope observations to quantify colonization rates. They demonstrated that DSEs had no effect on AMF colonization, while AMF colonization slightly decreased DSE frequency. We also monitored fluorescein diacetate (FDA) hydrolysis and alkaline phosphatase (AP) activity in the rhizosphere soils. FDA hydrolysis remained unchanged in the three inoculated treatments, but AMF colonization increased AP activity and P mobility in the soil whereas DSE colonization did not alter AP activity. In this experiment, we unveiled the interactions between two ecologically important fungal groups likely to occur in roots which involved a decrease of oxidative stress and Cd accumulation in shoots. These results open promising perspectives on the fungal-based phytomanagement of TE-contaminated sites by the production of uncontaminated and marketable plant biomass.