Death caps (Amanita phalloides) frequently establish from sexual spores, but individuals can grow large and live for more than a decade in invaded forests.

Global change is reshaping Earth's biodiversity, but the changing distributions of nonpathogenic fungi remain largely undocumented, as do mechanisms enabling invasions. The ectomycorrhizal Amanita phalloides is native to Europe and invasive in North America. Using population genetics and genomics, we sought to describe the life history traits of this successfully invading symbiotic fungus. To test whether death caps spread underground using hyphae, or aboveground using sexual spores, we mapped and genotyped mushrooms from European and US sites. Larger genetic individuals (genets) would suggest spread mediated by vegetative growth, while many small genets would suggest dispersal mediated by spores. To test whether genets are ephemeral or persistent, we also sampled from populations over time. At nearly every site and across all time points, mushrooms resolve into small genets. Individuals frequently establish from sexual spores. But at one Californian site, a single individual measuring nearly 10 m across dominated. At two Californian sites, the same genetic individuals were discovered in 2004, 2014, and 2015, suggesting single individuals (both large and small) can reproduce repeatedly over relatively long timescales. A flexible life history strategy combining both mycelial growth and spore dispersal appears to underpin the invasion of this deadly perennial ectomycorrhizal fungus.

Nitrogen fertilisation disrupts the temporal dynamics of arbuscular mycorrhizal fungal hyphae but not spore density and community composition in a wheat field.

Elucidating the temporal dynamics of arbuscular mycorrhizal (AM) fungi is critical for understanding their functions. Furthermore, research investigating the temporal dynamics of AM fungi in response to agricultural practices remains in its infancy. We investigated the effect of nitrogen fertilisation and watering reduction on the temporal dynamics of AM fungi, across the lifespan of wheat. Nitrogen fertilisation decreased AM fungal spore density (SD), extraradical hyphal density (ERHD), and intraradical colonisation rate (IRCR) in both watering conditions. Nitrogen fertilisation affected AM fungal community composition in soil but not in roots, regardless of watering conditions. The temporal analysis revealed that AM fungal ERHD and IRCR were higher under conventional watering and lower under reduced watering in March than in other growth stages at low (≤ 70 kg N ha-1 yr-1 ) but not at high (≥ 140) nitrogen fertilisation levels. AM fungal SD was lower in June than in other growth stages and community composition varied with plant development at all nitrogen fertilisation levels, regardless of watering conditions. This study demonstrates that high nitrogen fertilisation levels disrupt the temporal dynamics of AM fungal hyphal growth but not sporulation and community composition.

Fungal Seed Pathogens of Wild Chili Peppers Possess Multiple Mechanisms To Tolerate Capsaicinoids.

The wild chili pepper Capsicum chacoense produces the spicy defense compounds known as capsaicinoids, including capsaicin and dihydrocapsaicin, which are antagonistic to the growth of fungal pathogens. Compared to other microbes, fungi isolated from infected seeds of C. chacoense possess much higher levels of tolerance of these spicy compounds, having their growth slowed but not entirely inhibited. Previous research has shown capsaicinoids inhibit microbes by disrupting ATP production by binding NADH dehydrogenase in the electron transport chain (ETC) and, thus, throttling oxidative phosphorylation (OXPHOS). Capsaicinoids may also disrupt cell membranes. Here, we investigate capsaicinoid tolerance in fungal seed pathogens isolated from C. chacoense We selected 16 fungal isolates from four ascomycete genera (Alternaria, Colletotrichum, Fusarium, and Phomopsis). Using relative growth rate as a readout for tolerance, fungi were challenged with ETC inhibitors to infer whether fungi possess alternative respiratory enzymes and whether effects on the ETC fully explained inhibition by capsaicinoids. In all isolates, we found evidence for at least one alternative NADH dehydrogenase. In many isolates, we also found evidence for an alternative oxidase. These data suggest that wild-plant pathogens may be a rich source of alternative respiratory enzymes. We further demonstrate that these fungal isolates are capable of the breakdown of capsaicinoids. Finally, we determine that the OXPHOS theory may describe a weak primary mechanism by which dihydrocapsaicin, but not capsaicin, slows fungal growth. Our findings suggest that capsaicinoids likely disrupt membranes, in addition to energy poisoning, with implications for microbiology and human health.IMPORTANCE Plants make chemical compounds to protect themselves. For example, chili peppers produce the spicy compound capsaicin to inhibit pathogen damage and animal feeding. In humans, capsaicin binds to a membrane channel protein, creating the sensation of heat, while in microbes, capsaicin limits energy production by binding respiratory enzymes. However, some data suggest that capsaicin also disrupts membranes. Here, we studied fungal pathogens (Alternaria, Colletotrichum, Fusarium, and Phomopsis) isolated from a wild chili pepper, Capsicum chacoense By measuring growth rates in the presence of antibiotics with known respiratory targets, we inferred that wild-plant pathogens might be rich in alternative respiratory enzymes. A zone of clearance around the colonies, as well as liquid chromatography-mass spectrometry data, further indicated that these fungi can break down capsaicin. Finally, the total inhibitory effect of capsaicin was not fully explained by its effect on respiratory enzymes. Our findings lend credence to studies proposing that capsaicin may disrupt cell membranes, with implications for microbiology, as well as human health.

Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing.

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.

Effects of microplastics on soil properties: Current knowledge and future perspectives.

Microplastics (MPs) are a type of emerging contaminants that pose a potential threat to global terrestrial ecosystems, including agroecosystems. In recent years, MPs in soil and their adverse effects on soil health and fertility have attracted increasing concern. Based on the current knowledge, this review begins with a summary of the occurrence and characteristics of MPs in various soil environments, and then highlights the impacts of MPs on soil physical, chemical, and microbiological properties. Data show that MPs occur widely in all surveyed soil types, such as agricultural soils, industrial soils, urban soils, and unused soils, but show variation in their abundance, type, shape, and size. In most cases, MPs can change soil physical, chemical, and microbiological properties, but the effects vary, and are dependent on polymer type, shape, dose, and size. MPs-induced changes in soil fertility and the availability of pollutants may pose a potential threat to plant performance and crop productivity and safety. Particularly, MPs influence the emission of greenhouse gases from soil, ultimately leading to uncertain consequences for global climate change. More comprehensive and in-depth studies are required to fill large knowledge gaps.

Effects of Co-Contamination of Microplastics and Cd on Plant Growth and Cd Accumulation.

Microplastics (MPs) occur widely in terrestrial ecosystems. However, information on the interaction of MPs with metals in terrestrial ecosystems is lacking in the literature. The present study investigated the effects of two types of MPs (high-density polyethylene (HDPE) and polystyrene (PS)) with different dosages (i.e., 0, 0.1%, 1%, and 10%) on the uptake and effects of Cd in maize plants grown in an agricultural soil. Results showed that addition of Cd at a 5 mg/kg caused inhibited plant growth and resulted in high Cd accumulation in plant tissues. Polyethylene alone showed no significant phytotoxic effects, but a high-dose of HDPE (10%) amplified Cd phytotoxicity. Polystyrene negatively affected maize growth and phytoxicity further increased in the presence of Cd. Both HDPE and PS caused soil diethylenetriaminepentaacetic acid (DTPA)-extractable Cd concentrations to increase but did not significantly affect Cd uptake into plant tissues. In the soil without Cd addition, HDPE decreased soil pH, while PS did not significantly alter soil pH. However, in the soil spiked with Cd, both HDPE and PS increased pH. Overall, impacts on plant growth and Cd accumulation varied with MP type and dose, and PS induced substantial phytotoxicity. In conclusion, co-occurring MPs can change Cd bioavailability, plant performance, and soil traits. Our findings highlight the ecological impacts that could occur from the release of MPs into soil.

Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms.

The mushroom poison that causes the most deaths is the class of toxins known as amatoxins. Current methods to sensitively and selectively detect these toxins are limited by the need for expensive equipment, or they lack accuracy due to cross-reactivity with other chemicals found in mushrooms. In this work, we report the development of a competition-based lateral flow immunoassay (LFIA) for the rapid, portable, selective, and sensitive detection of amatoxins. Our assay clearly indicates the presence of 10 ng/mL of α-AMA or γ-AMA and the method including extraction and detection can be completed in approximately 10 minutes. The test can be easily read by eye and has a presumed shelf-life of at least 1 year. From testing 110 wild mushrooms, the LFIA identified 6 out of 6 species that were known to contain amatoxins. Other poisonous mushrooms known not to contain amatoxins tested negative by LFIA. This LFIA can be used to quickly identify amatoxin-containing mushrooms.

Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts.

Nuclear transfer stem cells hold considerable promise in the field of regenerative medicine and cell-based drug discovery. In this study, a total of 29 oocytes were obtained from three young (20-24 years old) reproductive egg donors who had been successful in previous cycles. These oocytes, deemed by intended parents to be in excess of their reproductive needs, were donated for research without financial compensation by both the egg donor and intended parents after receiving informed consent. All intended parents successfully achieved ongoing pregnancies with the oocytes retained for reproductive purposes. Mature oocytes, obtained within 2 hours following transvaginal aspiration, were enucleated using one of two methods, extrusion or aspiration, after 45 minutes of incubation in cytochalasin B. Rates of oocyte lysis or degeneration did not differ between the two methods. Somatic cell nuclear transfer (SCNT) embryos were constructed using two established adult male fibroblast lines of normal karyotype. High rates of pronuclear formation (66%), early cleavage (47%), and blastocyst (23%) development were observed following incubation in standard in vitro fertilization culture media. One cloned blastocyst was confirmed by DNA and mitochondrial DNA fingerprinting analyses, and DNA fingerprinting of two other cloned blastocysts indicated that they were also generated by SCNT. Blastocysts were also obtained from a limited number of parthenogenetically activated oocytes. This study demonstrates, for the first time, that SCNT can produce human blastocyst-stage embryos using nuclei obtained from differentiated adult cells and provides new information on methods that may be needed for a higher level of efficiency for human nuclear transfer.

Combined effects of ZnO NPs and Cd on sweet sorghum as influenced by an arbuscular mycorrhizal fungus.

Both metals and metal-based nanoparticles (NPs) can easily accumulate in soil, posing risks for plants and microbes. However, the interaction between NPs and toxic metals coexisting in soil is not yet well understood. Here, we studied the combined effects of ZnO NPs and Cd on sweet sorghum inoculated with or without the arbuscular mycorrhizal (AM) fungus Funneliformis caledonium. Plants were grown in soil amended with ZnO NPs (50, 250, and 500 mg/kg), alone or in combination with 5 mg/kg Cd. The two higher doses of ZnO NPs inhibited plant growth, leading to synergistic toxicity with Cd. However, at the lowest dose, ZnO NPs were non-phytotoxic, displaying antagonistic interactions with Cd on plant growth. When added with high doses of ZnO NPs, Cd significantly increased root Zn concentrations, but decreased shoot Zn concentrations at the low dose. Conversely, all doses of ZnO NPs significantly decreased shoot and root Cd concentrations. Furthermore, high doses of ZnO NPs generally inhibited soil enzyme activities, but Cd addition showed no significant or even stimulative effects, and mitigated the inhibitory effects of ZnO NPs. AM inoculation increased plant growth and P nutrition, and soil enzyme activities. When the low dose of ZnO NPs was added alone or in combination with Cd, AM inoculation decreased shoot Zn concentrations. Our results reveal complex interactions between ZnO NPs and Cd on plant growth and nutrition, plant Zn and Cd accumulation, and soil enzyme activities, while AM inoculation can help diminish the adverse effects induced by ZnO NPs and Cd.

Decreased ZnO nanoparticle phytotoxicity to maize by arbuscular mycorrhizal fungus and organic phosphorus.

ZnO nanoparticles (NPs) are applied in a wide variety of applications and frequently accumulate in the environment, thus posing risks to the environment and human health. Arbuscular mycorrhizal (AM) fungi (AMF) associate symbiotically with roots of most higher plants, helping their host plants acquire phosphorus (P). AMF can reduce the toxicity of ZnO NPs, but the benefits of AMF to host plants highly vary with soil available P. We hypothesize that organic P may help AMF to alleviate ZnO NP phytotoxicity. Here, we investigated the effects of inoculation with Funneliformis mosseae on plant growth and Zn accumulation, using maize grown in soil-sand mix substrates spiked with ZnO NPs (0 or 500 mg kg-1) under different organic P supply levels (0, 20, or 50 mg kg-1). The results showed addition of ZnO NPs inhibited root colonization rate, increased the shoot/root P concentration ratio, and led to significant Zn accumulation in soil and plants. As predicted, AM effects on maize plants all varied with P supply levels, both with or without ZnO NP additions. Organic P interacted synergistically with AMF to promote plant growth and acquisition of P, N, K, Fe, and Cu. AM inoculation reduced the bioavailable Zn released from ZnO NPs and decreased the concentrations and translocation of Zn to maize shoots. In conclusion, ZnO NPs caused excess Zn in soil and plants, posing potential environmental risks. However, our present results first demonstrate that organic P exhibited similar positive effects to AMF and interacted synergistically with AMF to improve plant growth and nutrition, and to decrease Zn accumulation and partitioning in plants, and thus helped diminish the adverse effects induced by ZnO NPs.

Pathogenic variant in NLRP7 (19q13.42) associated with recurrent gestational trophoblastic disease: Data from early embryo development observed during in vitro fertilization.

OBJECTIVE: To describe in vitro development of human embryos derived from an individual with a homozygous pathogenic variant in NLRP7 (19q13.42) and recurrent hydatidiform mole (HM), an autosomal recessive condition thought to occur secondary to an oocyte defect. METHODS: A patient with five consecutive HM pregnancies was genomically evaluated via next generation sequencing followed by controlled ovarian hyperstimulation, in vitro fertilization (IVF) with intracytoplasmic sperm injection, embryo culture, and preimplantation genetic screening. Findings in NLRP7 were recorded and embryo culture and biopsy data were tabulated as a function of parental origin for any identified ploidy error. RESULTS: The patient was found to have a pathogenic variant in NLRP7 (c.2810+2T>G) in a homozygous state. Fifteen oocytes were retrieved and 10 embryos were available after fertilization via intracytoplasmic sperm injection. Developmental arrest was noted for all 10 embryos after 144 hours in culture, thus no transfer was possible. These non-viable embryos were evaluated by karyomapping and all were diploid biparental; two were euploid and eight had various aneuploidies all of maternal origin. CONCLUSION: This is the first report of early human embryo development from a patient with any NLRP7 mutation. The pathogenic variant identified here resulted in global developmental arrest at or before blastocyst stage. Standard IVF should therefore be discouraged for such patients, who instead need to consider oocyte (or embryo) donation with IVF as preferred clinical methods to treat infertility.

Arbuscular mycorrhizae alleviate negative effects of zinc oxide nanoparticle and zinc accumulation in maize plants--A soil microcosm experiment.

ZnO nanoparticles (NPs) are considered an emerging contaminant when in high concentration, and their effects on crops and soil microorganisms pose new concerns and challenges. Arbuscular mycorrhizal (AM) fungi (AMF) form mutualistic symbioses with most vascular plants, and putatively contribute to reducing nanotoxicity in plants. Here, we studied the interactions between ZnO NPs and maize plants inoculated with or without AMF in ZnO NPs-spiked soil. ZnO NPs had no significant adverse effects at 400 mg/kg, but inhibited both maize growth and AM colonization at concentrations at and above 800 mg/kg. Sufficient addition of ZnO NPs decreased plant mineral nutrient acquisition, photosynthetic pigment concentrations, and root activity. Furthermore, ZnO NPs caused Zn concentrations in plants to increase in a dose-dependent pattern. As the ZnO NPs dose increased, we also found a positive correlation with soil diethylenetriaminepentaacetic acid (DTPA)-extractable Zn. However, AM inoculation significantly alleviated the negative effects induced by ZnO NPs: inoculated-plants experienced increased growth, nutrient uptake, photosynthetic pigment content, and SOD activity in leaves. Mycorrhizal plants also exhibited decreased ROS accumulation, Zn concentrations and bioconcentration factor (BCF), and lower soil DTPA-extractable Zn concentrations at high ZnO NPs doses. Our results demonstrate that, at high contamination levels, ZnO NPs cause toxicity to AM symbiosis, but AMF help alleviate ZnO NPs-induced phytotoxicity by decreasing Zn bioavailability and accumulation, Zn partitioning to shoots, and ROS production, and by increasing mineral nutrients and antioxidant capacity. AMF may play beneficial roles in alleviating the negative effects and environmental risks posed by ZnO NPs in agroecosystems.