Effects of the Dark Septate Endophyte (DSE) Exophiala pisciphila on the Growth of Root Cell Wall Polysaccharides and the Cadmium Content of Zea mays L. under Cadmium Stress

This paper aims to investigate the mechanism by which dark septate endophytes (DSEs) enhance cadmium (Cd) tolerance in there host plants. Maize (Zea mays L.) was inoculated with a DSE, Exophiala pisciphila, under Cd stress at different concentrations (0, 5, 10, and 20 mg·kg−1). The results show that, under 20 mg/kg Cd stress, DSE significantly increased maize biomass and plant height, indicating that DSE colonization can be utilized to increase the Cd tolerance of host plants. More Cd was retained in DSE-inoculated roots, especially that fixed in the root cell wall (RCW). The capability of DSE to induce a higher Cd holding capacity in the RCW is caused by modulation of the total sugar and uronic acid of DSE-colonized RCW, mainly the pectin and hemicellulose fractions. The fourier-transform spectroscopy analysis results show that carboxyl, hydroxyl, and acidic groups are involved in Cd retention in the DSE-inoculated RCW. The promotion of the growth of maize and improvement in its tolerance to Cd due to DSEs are related to restriction of the translocation of Cd from roots to shoots; resistance of Cd uptake Cd inside cells; and the increase in RCW-integrated Cd through modulating RCW polysaccharide components.

Metagenomic Analysis Reveals Reduced Beneficial Microorganism Associations in Roots of Foot Rot Affected Citrus Trees

Root endophytes have been shown to influence plant growth and plant responses to pathogens. Phytophthora spp. are important pathogens of citrus that cause foot rot and fibrous root decline. We hypothesize that foot rot infection by Phytophthora spp. can result in changes of the endophytic microbial community in citrus roots. Therefore, the endophytic (including bacterial and fungal) microbiome of roots from Phytophthora foot rot affected and healthy citrus trees were analyzed by Illumina sequencing of 16S rRNA and internal transcribed spacer (ITS) amplicons. Results indicate that the composition and structure of the endophytic bacterial and fungal communities were changed in roots of foot rot affected citrus trees. The populations of bacterial genera Asteroleplasma, Pseudomonas and Streptomyces were decreased in foot rot affected trees. Also, the relative abundance of Exophiala pisciphila and Glomeraceae spp. were significantly decreased in citrus roots. Pseudomonas and Streptomyces are considered beneficial bacteria and E. pisciphila and Glomeraceae spp. are dark septate endophytes and arbuscular mycorrhizal fungi respectively, which are involved in citrus health and growth. This study provides a baseline to continue investigating the interactions between the citrus host, Phytophthora spp. and beneficial microbes.

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.

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.

Melatonin Confers Heavy Metal Tolerance of a Dark Septate Endophyte (DSE) Exophiala Pisciphila Via Lowering Oxidative Stress and Heavy Metal Accumulation

Background: The high antioxidant capacity of melatonin contributing to heavy metal tolerance for plants and animals is widely studied, while researches on microorganisms especially in filamentous fungi are rare. One typical dark septate endophyte (DSE), Exophiala pisciphila, showed significant resistance to heavy metals.Results: In this study, exogenous melatonin was verified to reduce heavy metal damage via relieving oxidative stress, activating antioxidant systems, and decreasing heavy metal accumulation in E. pisciphila. Melatonin biosynthesis enzyme genes were upregulated under heavy metal stress. Furthermore, the overexpression of E. pisciphila TDC1 (EpTDC1) and E. pisciphila ASMT1 (EpASMT1) responsible for melatonin biosynthesis in Escherichia coli and Arabidopsis thaliana, enhanced heavy metal stress tolerance for the two organisms by lowering the oxidative stress and reducing the Cd accumulation in the whole plants, especially in the roots.Conclusions: Our results indicate that melatonin confers heavy metal resistance in E. pisciphila by lowering oxidative stress and heavy metal accumulation.

EpABC Genes in the Adaptive Responses of Exophiala pisciphila to Metal Stress: Functional Importance and Relation to Metal Tolerance

ABSTRACT Exophiala pisciphila is one of the dominant dark septate endophytes (DSEs) colonizing metal-polluted slag heaps in southwest China. It shows numerous super-metal-tolerant characteristics, but the molecular mechanisms involved remain largely unknown. In the present study, the functional roles of a specific set of ATP-binding cassette (ABC) transporters in E. pisciphila were characterized. In total, 26 EpABC genes belonging to 6 subfamilies (ABCA to ABCG) were annotated in previous transcriptome sequencing libraries, and all were regulated by metal ions (Pb, Zn, and Cd), which was dependent on the metal species and/or concentrations tested. The results from the heterologous expression of 3 representative EpABC genes confirmed that the expression of EpABC2.1, EpABC3.1, or EpABC4.1 restored the growth of metal-sensitive mutant Saccharomyces cerevisiae strains and significantly improved the tolerance of Arabidopsis thaliana to Pb, Zn, and Cd. Interestingly, the expression of the 3 EpABC genes further altered metal (Pb, Zn, and Cd) uptake and accumulation and promoted growth by alleviating the inhibitory activity in yeast and thale cress caused by toxic ions. These functions along with their vacuolar location suggest that the 3 EpABC transporters may enhance the detoxification of vacuolar compartmentation via transport activities across their membranes. In conclusion, the 26 annotated EpABC transporters may play a major role in maintaining the homeostasis of various metal ions in different cellular compartments, conferring an extreme adaptative advantage to E. pisciphila in metal-polluted slag heaps. IMPORTANCE Many ABC transporters and their functions have been identified in animals and plants. However, little is known about ABC genes in filamentous fungi, especially DSEs, which tend to dominantly colonize the roots of plants growing in stressed environments. Our results deepen the understanding of the function of the ABC genes of a super-metal-tolerant DSE (E. pisciphila) in enhancing its heavy metal resistance and detoxification. Furthermore, the genetic resources of DSEs, e.g., numerous EpABC genes, especially from super-metal-tolerant strains in heavy metal-polluted environments, can be directly used for transgenic applications to improve tolerance and phytoextraction potential.