Analysis of auxin responses in the fern Ceratopteris richardii identifies the developmental phase as a major determinant for response properties.

The auxin signaling molecule regulates a range of plant growth and developmental processes. The core transcriptional machinery responsible for auxin-mediated responses is conserved across all land plants. Genetic, physiological and molecular exploration in bryophyte and angiosperm model species have shown both qualitative and quantitative differences in auxin responses. Given the highly divergent ontogeny of the dominant gametophyte (bryophytes) and sporophyte (angiosperms) generations, however, it is unclear whether such differences derive from distinct phylogeny or ontogeny. Here, we address this question by comparing a range of physiological, developmental and molecular responses to auxin in both generations of the model fern Ceratopteris richardii. We find that auxin response in Ceratopteris gametophytes closely resembles that of a thalloid bryophyte, whereas the sporophyte mimics auxin response in flowering plants. This resemblance manifests both at the phenotypic and transcriptional levels. Furthermore, we show that disrupting auxin transport can lead to ectopic sporophyte induction on the gametophyte, suggesting a role for auxin in the alternation of generations. Our study thus identifies developmental phase, rather than phylogeny, as a major determinant of auxin response properties in land plants.

Non-seed plants are emerging gene sources for agriculture and insect control proteins.

The non-seed plants (e.g., charophyte algae, bryophytes, and ferns) have multiple human uses, but their contributions to agriculture and research have lagged behind seed plants. While sharing broadly conserved biology with seed plants and the major crops, non-seed plants sometimes possess alternative molecular and physiological adaptations. These adaptations may guide crop improvements. One such area is the presence of multiple classes of insecticidal proteins found in non-seed plant genomes which are either absent or widely diverged in seed plants. There are documented uses of non-seed plants, and ferns for example have been used in human diets. Among the occasional identifiable toxins or antinutritive components present in non-seed plants, none include these insecticidal proteins. Apart from these discrete risk factors which can be addressed in the safety assessment, there should be no general safety concern about sourcing genes from non-seed plant species.

The sperm nuclear basic proteins of the sword fern (Polystichum munitum).

Sperm nuclear basic proteins (SNBPs) were isolated from extracted antheridia-rich male gametophytes raised from spores of the swordfern, Polystichum munitum. Electrophoretic (acetic acid-urea PAGE and SDS-PAGE) and chromatographic (rp-HPLC) characterization of the nuclear proteins exhibited the characteristics of the histone (H-type). In both types of gel electrophoresis, histones H1, H2A, and H2B showed an altered electrophoretic mobility corresponding to that which is routinely observed for the histones in other plants. Histones present during spermatogenesis of the fern P. munitum were compared with the few current SNBPs known to be present in higher and lower evolutionary plant clades. A transition from an early protamine (P-type) SNBPs in charophytes and bryophytes to the (H-type) SNBP observed here is reminiscent of similar reversions observed in the animal kingdom.

Facultative mycorrhization in a fern (Struthiopteris spicant L. Weiss) is bound to light intensity.

BACKGROUND: The establishment of mycorrhizal relationships between a fungus and a plant typically enhances nutrient and water uptake for the latter while securing a carbon source for the fungus. However, under a particular set of environmental conditions, such as low availability of light and abundant nutrients in the soil, the resources invested in the maintenance of the fungi surpass the benefits obtained by the host. In those cases, facultative mycorrhizal plants are capable of surviving without symbiosis. Facultative mycorrhization in ferns has been overlooked until now. The present study measured the response of Struthiopteris spicant L. Weiss, and its root-associated fungi to different levels of light and nutrient availability in terms of growth, mycorrhizal presence, and leaf nutrient content. This fern species exhibits a great tolerance to variable light, nutrient, and pH conditions, and it has been found with and without mycorrhizae. We conducted a greenhouse experiment with 80 specimens of S. spicant and three factors (Light, Phosphorus, and Nitrogen) resulting in eight treatments. RESULTS: We found a significant influence of the factor light on fungal community composition, plant biomass, and nutrient accumulation. Departing from a lack of colonization at the initial stage, plants showed a remarkable increment of more than 80% in the arbuscular mycorrhizal fungi (AMF) richness and abundance in their roots when grown under high light conditions, compared with the ones in low light. We also observed an upward trend of C:P and C:N ratios and the above- and belowground biomass production when AMF abundance increased. Furthermore, the compositional analysis of the whole fungal communities associated with S. spicant roots revealed clear differences among low-light and high-light treatments. CONCLUSIONS: This study is the first to investigate the importance of light and nutrient availability in determining fern-AMF relationships. We confirmed that Struthiopteris spicant is a facultative mycorrhizal plant. The composition and diversity of AMF found in the roots of this fern are strongly influenced by light and less by nutrient conditions. Our study shows that ferns respond very sensitively to changes in environmental factors, leading to shifts in the associated mycorrhizal communities.

Transcriptomic analyses in the gametophytes of the apomictic fern Dryopteris affinis.

MAIN CONCLUSION: A novel genomic map of the apogamous gametophyte of the fern Dryopteris affinis unlocks oldest hindrance with this complex plant group, to gain insight into evo-devo approaches. The gametophyte of the fern Dryopteris affinis ssp. affinis represents a good model to explore the molecular basis of vegetative and reproductive development, as well as stress responses. Specifically, this fern reproduces asexually by apogamy, a peculiar case of apomixis whereby a sporophyte forms directly from a gametophytic cell without fertilization. Using RNA-sequencing approach, we have previously annotated more than 6000 transcripts. Here, we selected 100 of the inferred proteins homolog to those of Arabidopsis thaliana, which were particularly interesting for a detailed study of their potential functions, protein-protein interactions, and distance trees. As expected, a plethora of proteins associated with gametogenesis and embryogenesis in angiosperms, such as FERONIA (FER) and CHROMATING REMODELING 11 (CHR11) were identified, and more than a dozen candidates potentially involved in apomixis, such as ARGONAUTE family (AGO4, AGO9, and AGO 10), BABY BOOM (BBM), FASCIATED STEM4 (FAS4), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), and MATERNAL EFFECT EMBRYO ARREST29 (MEE29). In addition, proteins involved in the response to biotic and abiotic stresses were widely represented, as shown by the enrichment of heat-shock proteins. Using the String platform, the interactome revealed that most of the protein-protein interactions were predicted based on experimental, database, and text mining datasets, with MULTICOPY SUPPRESSOR OF IRA4 (MSI4) showing the highest number of interactions: 16. Lastly, some proteins were studied through distance trees by comparing alignments with respect to more distantly or closely related plant groups. This analysis identified DCL4 as the most distant protein to the predicted common ancestor. New genomic information in relation to gametophyte development, including apomictic reproduction, could expand our current vision of evo-devo approaches.

Polyploidy and environmental stress response: a comparative study of fern gametophytes.

Climate change is rapidly altering natural habitats and generating complex patterns of environmental stress. Ferns are major components of many forest understories and, given their independent gametophyte generation, may experience unique pressures in emerging temperature and drought regimes. Polyploidy is widespread in ferns and may provide a selective advantage in these rapidly changing environments. This work aimed to understand whether the gametophytes of allopolyploid ferns respond differently to climate-related physiological stress than their diploid parents. The experimental approach involved a multifactorial design with 27 treatment combinations including exposure to multiple levels of drought and temperature over three treatment durations, with recovery measured at multiple timepoints. We measured Chl fluorescence from over 2000 gametophytes to evaluate stress avoidance and tolerance in diploid and polyploid species. Polyploids generally showed a greater ability to avoid and/or tolerate a range of stress conditions compared with their diploid counterparts, suggesting that polyploidy may confer enhanced flexibility and resilience under climate stress. Overall, these results suggest that polyploidy may provide some resilience to climate change in mixed ploidy populations. However, all species remain susceptible to the impacts of extreme drought and heat stress.

Biosynthesis and differential spatial distribution of the 3-deoxyanthocyanidins apigenidin and luteolinidin at the interface of a plant-cyanobacteria symbiosis exposed to cold.

Aquatic ferns of the genus Azolla (Azolla) form highly productive symbioses with filamentous cyanobacteria fixing N2 in their leaf cavities, Nostoc azollae. Stressed symbioses characteristically turn red due to 3-deoxyanthocyanidin (DA) accumulation, rare in angiosperms and of unknown function. To understand DA accumulation upon cold acclimation and recovery, we integrated laser-desorption-ionization mass-spectrometry-imaging (LDI-MSI), a new Azolla filiculoides genome-assembly and annotation, and dual RNA-sequencing into phenotypic analyses of the symbioses. Azolla sp. Anzali recovered even when cold-induced DA-accumulation was inhibited by abscisic acid. Cyanobacterial filaments generally disappeared upon cold acclimation and Nostoc azollae transcript profiles were unlike those of resting stages formed in cold-resistant sporocarps, yet filaments re-appeared in leaf cavities of newly formed green fronds upon cold-recovery. The high transcript accumulation upon cold acclimation of AfDFR1 encoding a flavanone 4-reductase active in vitro suggested that the enzyme of the first step in the DA-pathway may regulate accumulation of DAs in different tissues. However, LDI-MSI highlighted the necessity to describe metabolite accumulation beyond class assignments as individual DA and caffeoylquinic acid metabolites accumulated differentially. For example, luteolinidin accumulated in epithelial cells, including those lining the leaf cavity, supporting a role for the former in the symbiotic interaction during cold acclimation.

Systematics and evolutionary dynamics of insect-fern interactions in the specialized fern-spore feeding Cuprininae (Lepidoptera, Stathmopodidae).

Fern-spore-feeding (FSF) is rare and found in only four families of Lepidoptera. Stathmopodidae is the most speciose family that contains FSF species, and its subfamily Cuprininae exclusively specializes on FSF. However, three species of Stathmopodinae also specialize on FSF. To better understand the evolutionary history of FSF and, more generally, the significance of specialization on a peculiar host, a phylogenetic and taxonomic revision for this group is necessary. We reconstructed the most comprehensive molecular phylogeny, including one mitochondrial and four nuclear genes, of Stathmopodidae to date, including 137 samples representing 62 species, with a particular focus on the FSF subfamily, Cuprininae, including 33 species (41% of named species) from 6 of the 7 Cuprininae genera. Species from two other subfamilies, Stathmopodinae and Atkinsoniinae, were also included. We found that FSF evolved only once in Stathmopodidae and that the previous hypothesis of multiple origins of FSF was misled by inadequate taxonomy. Moreover, we showed that (1) speciation/extinction rates do not differ significantly between FSF and non-FSF groups and that (2) oligophage is the ancestral character state in Cuprininae. We further revealed that a faster rate of accumulating specialists over time, and thus a higher number of specialists, was achieved by a higher transition rate from oligophagages to specialists compared to the transition rate in the opposite direction. We finish by describing three new genera, Trigonodagen. nov., Petalagen. nov., and Pediformisgen. nov., and revalidating five genera: Cuprina, Calicotis, Thylacosceles, Actinoscelis, Thylacosceloides in Cuprininae, and we provide an updated taxonomic key to genera and a revised global checklist of Cuprininae.

A phytochrome/phototropin chimeric photoreceptor promotes growth of fern gametophytes under limited light conditions.

Many ferns thrive even in low-light niches such as under an angiosperm forest canopy. However, the shade adaptation strategy of ferns is not well understood. Phytochrome 3/neochrome (phy3/neo) is an unconventional photoreceptor, found in the fern Adiantum capillus-veneris, that controls both red and blue light-dependent phototropism and chloroplast photorelocation, which are considered to improve photosynthetic efficiency in ferns. Here we show that phy3/neo localizes not only at the plasma membrane but also in the nucleus. Since both phototropism and chloroplast photorelocation are mediated by membrane-associated phototropin photoreceptors, we speculated that nucleus-localized phy3/neo possesses a previously undescribed biological function. We reveal that phy3/neo directly interacts with Adiantum cryptochrome 3 (cry3) in the nucleus. Plant cryptochromes are blue light receptors that transcriptionally regulate photomorphogenesis; therefore, phy3/neo may function via cry3 to synchronize light-mediated development with phototropism and chloroplast photorelocation to promote fern growth under low-light conditions. Furthermore, we demonstrate that phy3/neo regulates the expression of the Cyclin-like gene AcCyc1 and promotes prothallium expansion growth. These findings provide insight into the shade adaptation strategy of ferns and suggest that phy3/neo plays a substantial role in the survival and growth of ferns during the tiny gametophytic stage under low-light conditions, such as those on the forest floor.

All tangled up: Unraveling phylogenetics and reticulate evolution in the vining ferns, Lygodium (Schizaeales).

PREMISE: Reticulate evolution, often accompanied by polyploidy, is prevalent in plants, and particularly in the ferns. Resolving the resulting non-bifurcating histories remains a major challenge for plant phylogenetics. Here, we present a phylogenomic investigation into the complex evolutionary history of the vining ferns, Lygodium (Lygodiaceae, Schizaeales). METHODS: Using a targeted enrichment approach with the GoFlag 408 flagellate land plant probe set, we generated large nuclear and plastid sequence datasets for nearly all taxa in the genus and constructed the most comprehensive phylogeny of the family to date using concatenated maximum likelihood and coalescence approaches. We integrated this phylogeny with cytological and spore data to explore karyotype evolution and generate hypotheses about the origins of putative polyploids and hybrids. RESULTS: Our data and analyses support the origins of several putative allopolyploids (e.g., L. cubense, L. heterodoxum) and hybrids (e.g., L. ×fayae) and also highlight the potential prevalence of autopolyploidy in this clade (e.g., L. articulatum, L. flexuosum, and L. longifolium). CONCLUSIONS: Our robust phylogenetic framework provides valuable insights into dynamic reticulate evolution in this clade and demonstrates the utility of target-capture data for resolving these complex relationships.

Prevalent arbuscular mycorrhizae in roots and highly variable mycobiome in leaves of epiphytic subtropical fern Ophioderma pendulum.

PREMISE: Endophytic and mycorrhizal fungi are crucial in facilitating plant nutrition acquisition and stress tolerance. In epiphytic habitats, plants face nutrition and water stress, but their roots are mostly nonmycorrhizal and especially lacking in arbuscular mycorrhizal associations. Ophioderma pendulum is an epiphytic fern with a partially mycoheterotrophic lifestyle, likely heavily reliant on symbiotic fungi. To characterize fungal associations in the sporophyte of O. pendulum, we focused on leaves and roots of O. pendulum, seeking to reveal the fungal communities in these organs. METHODS: Roots and leaves from O. pendulum in a subtropical forest were examined microscopically to observe the morphology of fungal structures and determine the percentage of various fungal structures in host tissues. Fungal composition was profiled using metabarcoding techniques that targeted ITS2 of the nuclear ribosomal DNA. RESULTS: Roots were consistently colonized by arbuscular mycorrhizal fungi (Glomeromycota), especially Acaulospora. Unlike previous findings on epiphytic ferns, dark septate endophytes were rare in O. pendulum roots. Leaves were predominantly colonized by Ascomycota fungi, specifically the classes Dothideomycetes (46.88%), Eurotiomycetes (11.51%), Sordariomycetes (6.23%), and Leotiomycetes (6.14%). Across sampling sites, fungal community compositions were similar in the roots but differed significantly in the leaves. CONCLUSIONS: Ophioderma pendulum maintains stable, single-taxon-dominant communities in the roots, primarily featuring arbuscular mycorrhizal fungi, whereas the leaves may harbor opportunistic fungal colonizers. Our study underlines the significance of mycorrhizal fungi in the adaptation of epiphytic ferns.

The non-flowering plants of a near-polar forest in East Gondwana, Tasmania, Australia, during the Early Eocene Climatic Optimum.

PREMISE: The Cenozoic Macquarie Harbour Formation (MHF) hosts one of the oldest and southernmost post-Cretaceous fossil plant assemblages in Australia. Coinciding with the Early Eocene Climatic Optimum (EECO) and predating the breakup of Australia from Antarctica, it offers critical data to study the diversity and extent of the Austral Polar Forest Biome, and the floristic divergence between Australasia and South America resulting from the Gondwana breakup. METHODS: The micromorphology and macromorphology of new fossil plant compressions from the MHF were described and systematically analyzed. Previously published non-flowering plant records were reviewed and revised. Macrofossil abundance data were provided. The flora was compared with other early Paleogene assemblages from across the Southern Hemisphere. RESULTS: Twelve species of non-flowering plants were identified from the macrofossil record. Conifers include Araucariaceae (Araucaria macrophylla, A. readiae, A. timkarikensis sp. nov., and Araucarioides linearis), Podocarpaceae (Acmopyle glabra, Dacrycarpus mucronatus, Podocarpus paralungatikensis sp. nov., and Retrophyllum sp.), and Cupressaceae (Libocedrus microformis). Dacrycarpus linifolius was designated a junior synonym of D. mucronatus. Further components include a cycad (Bowenia johnsonii, Zamiaceae), a pteridosperm (Komlopteris cenozoicus, Umkomasiaceae), and a fern (Lygodium dinmorphyllum, Schizaeaceae). CONCLUSIONS: The fossil assemblage represents a mixed near-polar forest with a high diversity of conifers. The morphology and preservation of several species indicate adaptations to life at high latitudes. The coexistence of large- and small-leaved conifers implies complex, possibly open forest structures. Comparisons with contemporaneous assemblages from Argentina support a circumpolar biome during the EECO, reaching from southern Australia across Antarctica to southern South America.

Linking the macroclimatic niche of native lithophytic ferns and their prevalence in urban environments.

PREMISE: Vertical surfaces in urban environments represent a potential expansion of niche space for lithophytic fern species. There are, however, few records of differential success rates of fern species in urban environments. METHODS: The occurrence rates of 16 lithophytic fern species native to the northeastern USA in 14 biomes, including four urban environments differentiated by percentage of impervious surfaces, were evaluated. In addition, the natural macroclimatic ranges of these species were analyzed to test whether significant differences existed in climatic tolerance between species that occur in urban environments and species that do not. RESULTS: Three species appear to preferentially occur in urban environments, two species may facultatively occur in urban environments, and the remaining 11 species preferentially occur in nondeveloped rural environments. The natural range of fern species that occur in urban environments had higher summer temperatures than the range of species that do not, whereas other macroclimatic variables, notably winter temperatures and precipitation, were less important or insignificant. CONCLUSIONS: Vertical surfaces in urban environments may represent novel niche space for some native lithophytic fern species in northeastern USA. However, success in this environment depends, in part, on tolerance of the urban heat island effect, especially heating of impervious surfaces in summer.

An adventurous journey toward and away from fern apomixis: Insights from genome size and spore abortion patterns.

PREMISE: Apomixis in ferns is relatively common and obligatory. Sterile hybrids may restore fertility via apomixis at a cost of long-term genetic stagnation. In this study, we outlined apomixis as a possible temporary phase leading to sexuality and analyzed factors relating to transitioning to and away from apomixis, such as unreduced and reduced spore formation in apomict and apo-sex hybrid ferns. METHODS: We analyzed the genome size of 15 fern species or hybrids ("taxa") via flow cytometry. The number of reduced and unreduced gametophytes was established as a proxy for viable spore formation of either type. We also calculated the spore abortion ratio (sign of reduced spores) in several taxa, including the apo-sex hybrid Dryopteris × critica and its 16 apomictically formed offspring. RESULTS: Four of 15 sampled taxa yielded offspring variable in genome size. Specifically, each variable taxon formed one viable reduced plant among 12-451 sampled gametophytes per taxon. Thus, haploid spore formation in the studied apomicts was very rare but possible. Spore abortion analyses indicated gradually decreasing abortion (haploid spore formation) over time. In Dryopteris × critica, abortion decreased from 93.8% to mean 89.5% in one generation. CONCLUSIONS: Our results support apomixis as a transitionary phase toward sexuality. Newly formed apomicts hybridize with sexual relatives and continue to form haploid spores early on. Thus, they may get the genomic content necessary for regular meiosis and restore sexuality. If the missing relative goes extinct, the lineage gets locked into apomixis as may be the case with the Dryopteris affinis complex.

Coordination of pinna, petiole, and root anatomical traits in 24 tropical-subtropical fern species.

Ferns are primitive vascular plants with diverse morphologies and structures. Plant anatomical traits and their linkages can reflect adaptation to the environment; however, these remain are still poorly understood in ferns. The main objective of this study was to explore whether there was structural coordination among and within organs in fern species. We measured 16 hydraulically related anatomical traits of pinnae, petioles, and roots of 24 representative fern species from the tropical and subtropical forest understory and analyzed trait correlation networks. In addition, we examined phylogenetic signals for the anatomical traits and analyzed co-evolutionary relationships. These results indicated that stomatal density and all petiole anatomical traits exhibited significant phylogenetic signals. Evolutionary correlations were observed between the tracheid diameter and wall thickness of the petiole and between the water transport capacity of the petiole and stomatal density. Conversely, anatomical traits of roots (e.g., root diameter) showed no phylogenetic signals and were not significantly correlated with those of the pinnae and petioles, indicating a lack of structural coordination between the below- and above-ground organs. Unlike angiosperms, vein density is unrelated to stomatal density or pinna thickness in ferns. As root diameter decreased, the cortex-to-stele diameter ratio decreased significantly (enhanced water absorption) in angiosperms but remained unchanged in ferns. These differences lead to different responses of ferns to climate change and improve our knowledge of the water adaptation strategies of ferns.

2,4-D mediated moderation of aluminum tolerance in Salvinia molesta D. Mitch. with regards to bioexclusion and related physiological and metabolic changes.

We examined the efficacy of 2,4-dichlorophenoxy acetic acid (2,4-D; 500 µM) in enhancing the potential of Salvinia species for tolerance to aluminum (Al) toxicity (240 and 480 µM, seven days). Salvinia showed better efficacy in removal of toxicity of Al by sorption mechanism with changes of bond energy shifting on cell wall residues and surface structure. Plants recorded tolerance to Al concentration (480 µM) when pretreated with 2,4-D through adjustment of relative water content, proline content, osmotic potential, and improved the pigment fluorescence for energy utilization under Al stress. Photosynthetic activities with regards to NADP-malic enzyme and malic dehydrogenase and sugar metabolism with wall and cytosolic invertase activities were strongly correlated with compatible solutes. A less membrane peroxidation and protein carbonylation had reduced ionic loss over the membrane that was studied with reduced electrolyte leakage with 2,4-D pretreated plants. Membrane stabilization was also recorded with higher ratio of K+ to Na+, thereby suggesting roles of 2,4-D in ionic balance. Better sustenance of enzymatic antioxidation with peroxidase and glutathione metabolism reduced reactive oxygen species accumulation and save the plant for oxidative damages. Moreover, gene polymorphism for antioxidant, induced by 2,4-D varied through Al concentrations would suggest an improved biomarker for tolerance. Collectively, analysis and discussion of plant's responses assumed that auxin herbicide could be a potential phytoprotectant for Salvinia as well as improving the stability to Al toxicity and its bioremediation efficacy.

In previous reports, aquatic weeds, particularly, from pteridophytic flora have been exercised, however, in less frequent. Aluminum (Al) toxicity, being a major problem, specifically with respect to cultivated crops like rice and vegetables, is a serious issue in alkaline soil. In context to growth of Salvinia in the areas of low lands where few important crops like rice are frequently cultivated. Therefore, Al toxicity with regards to rice cultivation in low land conditions, which is habitat for Salvinia, could be interesting. Thus, decontamination of low land for salinity with aquatic environment can be remediated with biological materials where Salvinia would be a choice. This would be something new in studies for the aquatic weeds over the existing database. Moreover, 2,4-dichlorophenoxy acetic acid (2,4-D) being a common herbicide in agricultural field that becomes more problematic with metal toxicity is another focus for physiological responses with Salvinia. The adoption and sustainability of Salvinia against 2,4-D may highlight insights for physiological activities would be the biomarker for herbicide toxicity.

Rapid Detection of the Strawberry Foliar Nematode Aphelenchoides fragariae Using Recombinase Polymerase Amplification Assay with Lateral Flow Dipsticks.

Rapid and reliable diagnostic methods for plant-parasitic nematodes are critical for facilitating the selection of effective control measures. A diagnostic recombinase polymerase amplification (RPA) assay for Aphelenchoides fragariae using a TwistAmp® Basic Kit (TwistDx, Cambridge, UK) and AmplifyRP® Acceler8® Discovery Kit (Agdia, Elkhart, IN, USA) combined with lateral flow dipsticks (LF) has been developed. In this study, a LF-RPA assay was designed that targets the ITS rRNA gene of A. fragariae. This assay enables the specific detection of A. fragariae from crude nematode extracts without a DNA extraction step, and from DNA extracts of plant tissues infected with this nematode species. The LF-RPA assay showed reliable detection within 18-25 min with a sensitivity of 0.03 nematode per reaction tube for crude nematode extracts or 0.3 nematode per reaction tube using plant DNA extracts from 0.1 g of fresh leaves. The LF-RPA assay was developed and validated with a wide range of nematode and plant samples. Aphelenchoides fragariae was identified from seed samples in California. The LF-RPA assay has great potential for nematode diagnostics in the laboratory with minimal available equipment.

Cytotoxic Natural Products from the Jurassic Relict Osmunda regalis L.

The Jurassic relict Royal fern, Osmunda regalis L., is widely distributed across temperate zones in the Northern and Southern hemispheres. Even though this species has been utilised for centuries as a medicinal plant, its phytochemical composition mainly remains unknown. As part of our ongoing research to identify potential lead compounds for future anticancer drugs, 17 natural products were characterised from the aerial parts of Osmunda regalis L. Fifteen of these compounds were identified in this species for the first time, including the six previously undescribed compounds kaempferol 3-O-(2''-O-(2'''-α-rhamnopyranosyl)-ß-glucopyranosyl)-ß-glucopyranoside, quercetin 3-O-(2''-O-(2'''-α-rhamnopyranosyl)-ß-glucopyranosyl)-ß-glucopyranoside, kaempferol 3-O-(2''-O-(2'''-α-rhamnopyranosyl-6'''-O-(E)-caffeoyl-)-ß-glucopyranosyl)-ß-glucopyranoside, 3-methoxy-5-hydroxy-4-olide, 4-hydroxy-3-(3'-hydroxy-4'-(hydroxyethyl)-oxotetrafuranone-5-methyl tetrahydropyranone, and 4-O-(5-hydroxy-4-oxohexanoyl) osmundalactone. The molecular structures were determined by combining several 1D and 2D NMR experiments, circular dichroism spectroscopy, and HRMS. Determination of cytotoxicity against AML MOLM-13, H9c2, and NRK cell lines showed that two isolated lactones exhibited significant cytotoxic activity.

Preparation of fern root resistant starch by pullulanase and glucoamylase combined with autoclaving-enzymatic method: physicochemical properties and structural characterization.

BACKGROUND: Fern root starch has a high percentage of amylose and has great potential for application in the field of slow-digesting foods. Clarifying the effect of treatment conditions on fern root starch is key to achieving industrialized production of fern root resistant starch. In the present study, fern root starch was treated by the autoclave-enzymatic method with pullulanase, glucoamylase and mixed enzyme. RESULTS: The content of resistant starch in fern roots treated with mixed enzyme was the highest (24.07 ± 1.11%), which was approximately 320% times that of the native starch, had the best water-holding capacity (151.08%), vital transparency and freeze-thaw stability. By contrast, the solubility, swelling and viscosity were lower than natural starch. In addition, mixed enzyme shows a denser structure, and the crystal form changes from C-type to V-type, with a high relative crystallinity and significantly enhanced thermal stability. CONCLUSION: After mixed enzyme combined with autoclave treatment, the content of resistant starch in fern root was greatly increased. The modified starch molecules did not produce new functional groups, which made the crystal structure of starch molecules more compact, and resistance to enzymatic hydrolysis and high temperature thermal stability were significantly enhanced. This provides a positive reference for further in-depth study of fern root starch, improvement of utilization value, development and innovation of new food health products, and diabetes treatment. © 2024 Society of Chemical Industry.

Stable coexistence or competitive exclusion? Fern endophytes demonstrate rapid turnover favouring a dominant fungus.

Fungal endophytes are critical members of the plant microbiome, but their community dynamics throughout an entire growing season are underexplored. Additionally, most fungal endophyte research has centred on seed-reproducing hosts, while spore-reproducing plants also host endophytes and may be colonized by unique community members. In order to examine annual fungal endophyte community dynamics in a spore-reproducing host, we explored endophytes in a single population of ferns, Polystichum munitum, in the Pacific Northwest. Through metabarcoding, we characterized the community assembly and temporal turnover of foliar endophytes throughout a growing season. From these results, we selected endophytes with outsized representations in sequence data and performed in vitro competition assays. Finally, we inoculated sterile fern gametophytes with dominant fungi observed in the field and determined their effects on host performance. Sequencing demonstrated that ferns were colonized by a diverse community of fungal endophytes in newly emerged tissue, but diversity decreased throughout the season leading to the preponderance of a single fungus in later sampling months. This previously undescribed endophyte appears to abundantly colonize the host to the detriment of other microfungi. Competition assays on a variety of media types failed to demonstrate that the dominant fungus was competitive against other fungi isolated from the same hosts, and inoculation onto sterile fern gametophytes did not alter growth compared to sterile controls, suggesting its effects are not antagonistic. The presence of this endophyte in the fern population probably demonstrates a case of repeated colonization driving competitive exclusion of other fungal community members.