Functional Characterization of Ao4g24: An Uncharacterized Gene Involved in Conidiation, Trap Formation, Stress Response, and Secondary Metabolism in Arthrobotrys oligospora.

Arthrobotrys oligospora is a typical nematode-trapping (NT) fungus, which can secrete food cues to lure, capture, and digest nematodes by triggering the production of adhesive networks (traps). Based on genomic and proteomic analyses, multiple pathogenic genes and proteins involved in trap formation have been characterized; however, there are numerous uncharacterized genes that play important roles in trap formation. The functional studies of these unknown genes are helpful in systematically elucidating the complex interactions between A. oligospora and nematode hosts. In this study, we screened the gene AOL_s00004g24 (Ao4g24). This gene is similar to the SWI/SNF chromatin remodeling complex, which was found to play a potential role in trap formation in our previous transcriptome analysis. Here, we characterized the function of Ao4g24 by gene disruption, phenotypic analysis, and metabolomics. The deletion of Ao4g24 led to a remarkable decrease in conidia yield, trap formation, and secondary metabolites. Meanwhile, the absence of Ao4g24 influenced the mitochondrial membrane potential, ATP content, autophagy, ROS level, and stress response. These results indicate that Ao4g24 has crucial functions in sporulation, trap formation, and pathogenicity in NT fungi. Our study provides a reference for understanding the role of unidentified genes in mycelium growth and trap formation in NT fungi.

Nematode-trapping fungus Arthrobotrys oligospora recruited rhizosphere microorganisms to cooperate in controlling root-knot nematodes in tomato.

AIMS: The objective of this study was to elucidate the role and mechanism of changes in the rhizosphere microbiome following Arthrobotrys oligospora treatment in the biological control of root-knot nematodes and identify the key fungal and bacterial species that collaborate with A. oligospora to biocontrol root-knot nematodes. METHODS AND RESULTS: We conducted a pot experiment to investigate the impact of A. oligospora treatment on the biocontrol efficiency of A. oligospora against Meloidogyne incognita infecting tomatoes. We analyzed the rhizosphere bacteria and fungi communities of tomato by high-throughput sequencing of the 16S rRNA gene fragment and the internal transcribed spacer (ITS). The results indicated that the application of A. oligospora resulted in a 53.6% reduction in the disease index of M. incognita infecting tomato plants. The bacterial diversity of rhizosphere soil declined in the A. oligospora-treated group, while fungal diversity increased. The A. oligospora treatment enriched the tomato rhizosphere with Acidobacteriota, Firmicutes, Bradyrhizobium, Sphingomonadales, Glomeromycota, and Purpureocillium. These organisms are involved in the utilization of rhizosphere organic matter, nitrogen, and glycerolipids, or play the role of ectomycorrhiza or directly kill nematodes. The networks of bacterial and fungal co-occurrence exhibited a greater degree of stability and complexity in the A. oligospora treatment group. CONCLUSIONS: This study demonstrated the key fungal and bacterial species that collaborate with the A. oligospora in controlling the root-knot nematode and elaborated the potential mechanisms involved. The findings offer valuable insights and inspiration for the advancement of bionematicide based on nematode-trapping fungi.

Linking watershed formation with the phylogenetic distribution of a soil microscopic fungus in Yunnan Province, China.

BACKGROUND: Phylogeographic studies have gained prominence in linking past geological events to the distribution patterns of biodiversity, primarily in mountainous regions. However, such studies often focus on plant taxa, neglecting the intricate biogeographical patterns of microbes, particularly soil microbial communities. This article explores the spatial distribution of the nematode-trapping fungus Arthrobotrys oligospora, a widespread microorganism, in a tectonically active region at the southeastern edge of the Qinghai-Tibetan Plateau. By analysing the genetic variation of this fungus alongside the historical structure of major river watersheds, we sought to uncover potential connections between the two. Our study involved sampling 149 strains from 116 sites across six major watersheds in the region. RESULTS: The resulting haplotype network revealed five distinct clusters, each corresponding closely to a specific watershed. These clusters exhibited high haplotype diversity and low nucleotide diversity, supporting the notion of watershed-based segregation. Further analysis of haplotypes shared across watersheds provided evidence for three proposed past river connections. In particular, we found numerous shared haplotypes between the Yangtze and Mekong basins, as well as between the Yangtze and the Red basins. Evidence for a Irrawaddy-Salween-Red and a Yangtze-Pearl-Red river connections were also portrayed in our mapping exercise. CONCLUSIONS: These findings emphasize the crucial role of historical geomorphological events in shaping the biogeography of microbial biodiversity, alongside contemporary biotic and abiotic factors. Watershed perimeters emerged as effective predictors of such patterns, suggesting their suitability as analytical units for regional-scale studies. Our study also demonstrates the potential of microorganisms and phylogeographic approaches to complement traditional geological analyses, providing a more comprehensive understanding of past landscape structure and its evolution.

Characterization of genome-wide phylogenetic conflict uncovers evolutionary modes of carnivorous fungi.

Mass extinction has often paved the way for rapid evolutionary radiation, resulting in the emergence of diverse taxa within specific lineages. The emergence and diversification of carnivorous nematode-trapping fungi (NTF) in Ascomycota have been linked to the Permian-Triassic (PT) extinction, but the processes underlying NTF radiation remain unclear. We conducted phylogenomic analyses using 23 genomes that represent three NTF lineages, each employing distinct nematode traps-mechanical traps (Drechslerella spp.), three-dimensional (3D) adhesive traps (Arthrobotrys spp.), and two-dimensional (2D) adhesive traps (Dactylellina spp.), and the genome of one non-NTF species as the outgroup. These analyses revealed multiple mechanisms that likely contributed to the tempo of the NTF evolution and rapid radiation. The species tree of NTFs based on 2,944 single-copy orthologous genes suggested that Drechslerella emerged earlier than Arthrobotrys and Dactylellina. Extensive genome-wide phylogenetic discordance was observed, mainly due to incomplete lineage sorting (ILS) between lineages. Two modes of non-vertical evolution (introgression and horizontal gene transfer) also contributed to phylogenetic discordance. The ILS genes that are associated with hyphal growth and trap morphogenesis (e.g., those associated with the cell membrane system and polarized cell division) exhibited signs of positive selection.IMPORTANCEBy conducting a comprehensive phylogenomic analysis of 23 genomes across three NTF lineages, the research reveals how diverse evolutionary mechanisms, including ILS and non-vertical evolution (introgression and horizontal gene transfer), contribute to the swift diversification of NTFs. These findings highlight the complex evolutionary dynamics that drive the rapid radiation of NTFs, providing valuable insights into the processes underlying their diversity and adaptation.

Low microbial diversity, yeast prevalence, and nematode-trapping fungal presence in fungal colonization and leaf microbiome of Serjania erecta.

Medicinal plant microbiomes undergo selection due to secondary metabolite presence. Resident endophytic/epiphytic microorganisms directly influence plant's bioactive compound synthesis. Hypothesizing low microbial diversity in Serjania erecta leaves, we assessed leaf colonization by epiphytic and endophytic fungi. Given its traditional medicinal importance, we estimated diversity in the endophytic fungal microbiome. Analyses included scanning electron microscopy (SEM), isolation of cultivable species, and metagenomics. Epiphytic fungi interacted with S. erecta leaf tissues, horizontally transmitted via stomata/trichome bases, expressing traits for nematode trapping. Cultivable endophytic fungi, known for phytopathogenic habits, didn't induce dysbiosis symptoms. This study confirms low leaf microbiome diversity in S. erecta, with a tendency towards more fungal species, likely due to antibacterial secondary metabolite selection. The classification of Halicephalobus sp. sequence corroborated the presence of nematode eggs on the epidermal surface of S. erecta by SEM. In addition, we confirmed the presence of methanogenic archaea and a considerable number of methanotrophs of the genus Methylobacterium. The metagenomic study of endophytic fungi highlighted plant growth-promoting yeasts, mainly Malassezia, Leucosporidium, Meyerozyma, and Hannaella. Studying endophytic fungi and S. erecta microbiomes can elucidate their impact on beneficial bioactive compound production, on the other hand, it is possible that the bioactive compounds produced by this plant can recruit specific microorganisms, impacting the biological system.

Transcriptome analysis highlights the influence of temperature on hydrolase and traps in nematode-trapping fungi.

Pine wilt disease caused by Bursaphelenchus xylophilus poses a serious threat to the economic and ecological value of forestry. Nematode trapping fungi trap and kill nematodes using specialized trapping devices, which are highly efficient and non-toxic to the environment, and are very promising for use as biological control agents. In this study, we isolated several nematode-trapping fungi from various regions and screened three for their high nematocidal efficiency. However, the effectiveness of these fungi as nematicides is notably influenced by temperature and exhibits different morphologies in response to temperature fluctuations, which are categorized as "NA," "thin," "dense," and "sparse." The trend of trap formation with temperature was consistent with the trend of nematocidal efficiency with temperature. Both of which initially increased and then decreased with increasing temperature. Among them, Arthrobotrys cladodes exhibited the highest level of nematocidal activity and trap formation among the tested species. Transcriptome data were collected from A. cladodes with various trap morphologies. Hydrolase activity was significantly enriched according to GO and KEGG enrichment analyses. Eight genes related to hydrolases were found to be consistent with the trend of trap morphology with temperature. Weighted gene co-expression analysis and the Cytoscape network revealed that these 8 genes are associated with either mitosis or autophagy. This suggests that they contribute to the formation of "dense" structures in nematode-trapping fungi. One of these genes is the serine protein hydrolase gene involved in autophagy. This study reveals a potentially critical role for hydrolases in trap formation and nematocidal efficiency. And presents a model where temperature affects trap formation and nematocidal efficiency by influencing the serine protease prb1 involved in the autophagy process.

Endosymbiotic bacteria within the nematode-trapping fungus Arthrobotrys musiformis and their potential roles in nitrogen cycling.

Endosymbiotic bacteria (ESB) have important effects on their hosts, contributing to its growth, reproduction and biological functions. Although the effects of exogenous bacteria on the trap formation of nematode-trapping fungi (NTF) have been revealed, the effects of ESB on NTF remain unknown. In this study, we investigated the species diversity of ESB in the NTF Arthrobotrys musiformis using high-throughput sequencing and culture-dependent approaches, and compared bacterial profiles to assess the effects of strain source and culture media on A. musiformis. PICRUSt2 and FAPROTAX were used to predict bacterial function. Our study revealed that bacterial communities in A. musiformis displayed high diversity and heterogeneity, with Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria as the dominant phyla. The ESB between A. musiformis groups isolated from different habitats and cultured in the same medium were more similar to each other than the other groups isolated from the same habitat but cultured in different media. Function analysis predicted a broad and diverse functional repertoire of ESB in A. musiformis, and unveiled that ESB have the potential to function in five modules of the nitrogen metabolism. We isolated nitrogen-fixing and denitrifying bacteria from the ESB and demonstrated their effects on trap formation of A. musiformis. Among seven bacteria that we tested, three bacterial species Bacillus licheniformis, Achromobacter xylosoxidans and Stenotrophomonas maltophilia were found to be efficient in inducing trap formation. In conclusion, this study revealed extensive ESB diversity within NTF and demonstrated that these bacteria likely play important roles in nitrogen cycling, including nematode trap formation.

Secondary Metabolites from the Nematode-Trapping Fungus Dactylellina haptotyla YMF1.03409.

As a representative nematode-trapping fungus, Dactylellina haptotyla can capture and kill nematodes by producing traps, known as adhesive knobs. In this paper, the strain of D. haptotyla YMF1.03409 was studied by means of medium screening, fermentation, and purification and identification of crude extracts. Eighteen compounds were obtained from D. haptotyla YMF1.03409, including two new metabolites, nosporins C (1) and D (2). The known metabolites were identified to be 3-chloro-4-methoxybenzaldehyde (3), 3-chloro-4-methoxybenzoic acid (4), 2-chloro-1-methoxy-4-(methoxymethyl)benzene (5), 3-hydroxy-3-methyloxindole (6), nicotinic acid (7), succinic acid (8), 3,4-dihydroxybutanoic acid (9), 5'-O-methyladenosine (10), uridine (11), 2'-deoxyuridine (12), thymidine (13), 3-(phenylmethyl)-2,5-morpholinedione (14), methyl-ß-D-glucopyranoside (15), 1,2-benzenedicarboxylic acid bis(2-methyl heptyl) ester (16), ß-sitosterol (17), and 3ß,6α-diol-stigmastane (18). The bioactive assay showed that these compounds had no obvious nematicidal activity against the nematodes Meloidogyne incognita and Panagrellus redivivus.

2-Furoic acid associated with the infection of nematodes by Dactylellina haptotyla and its biocontrol potential on plant root-knot nematodes.

Dactylellina haptotyla is a typical nematode-trapping fungus that has garnered the attention of many scholars for its highly effective lethal potential for nematodes. Secondary metabolites play an important role in D. haptotyla-nematode interactions, but which metabolites perform which function remains unclear. We report the metabolic functions based on high-quality, chromosome-level genome assembly of wild D. haptotyla YMF1.03409. The results indicate that a large variety of secondary metabolites and their biosynthetic genes were significantly upregulated during the nematode-trapping stage. In parallel, we identified that 2-furoic acid was specifically produced during nematode trapping by D. haptotyla YMF1.03409 and isolated it from fermentation production. 2-Furoic acid demonstrated strong nematicidal activity with an LD50 value of 55.05 µg/mL against Meloidogyne incognita at 48 h. Furthermore, the pot experiment showed that the number of galls of tomato root was significantly reduced in the experimental group treated with 2-furoic acid. The considerable increase in the 2-furoic acid content during the infection process and its virulent nematicidal activity revealed an essential synergistic effect during the process of nematode-trapping fungal infection. IMPORTANCE Dactylellina haptotyla have significant application potential in nematode biocontrol. In this study, we determined the chromosome-level genome sequence of D. haptotyla YMF1.03409 by long-read sequencing technology. Comparative genomic analysis identified a series of pathogenesis-related genes and revealed significant gene family contraction events during the evolution of D. haptotyla YMF1.03409. Combining transcriptomic and metabolomic data as well as in vitro activity test results, a compound with important application potential in nematode biocontrol, 2-furoic acid, was identified. Our result expanded the genetic resource of D. haptotyla and identified a previously unreported nematicidal small molecule, which provides new options for the development of plant biocontrol agents.

Conidia Fusion: A Mechanism for Fungal Adaptation to Nutrient-Poor Habitats.

Conidia fusion (CF) is a commonly observed structure in fungi. However, it has not been systematically studied. This study examined 2457 strains of nematode-trapping fungi (NTF) to explore the species specificity, physiological period, and physiological significance of CF. The results demonstrated that only six species of Arthrobotrys can form CF among the sixty-five tested NTF species. The studies on the model species Arthrobotrys oligospora (DL228) showed that CF occurred in both shed and unshed plus mature and immature conidia. Additionally, the conidia fusion rate (CFR) increased significantly with the decrease of nutrient concentration in habitats. The studies on the conidia fusion body (CFB) produced by A. oligospora (DL228) revealed that the more conidia contained in the CFB, the faster and denser the mycelia of the CFB germinated in weak nutrient medium and soil plates. On the one hand, rapid mycelial extension is beneficial for the CFB to quickly find new nutrient sources in habitats with uneven nutrient distribution. On the other hand, dense mycelium increases the contact area with the environment, improving the nutrient absorption efficiency, which is conducive to improving the survival rate of conidia in the weak nutrient environment. In addition, all species that form CF produce smaller conidia. Based on this observation, CF may be a strategy to balance the defects (nutrient deficiency) caused by conidia miniaturization.

Function discovery of a non-ribosomal peptide synthetase-like encoding gene in the nematode-trapping fungus Arthrobotrys oligospora.

In this study, the function of a non-ribosomal peptide synthetase-like (NRPS-like) encoding gene AOL_s00188g306 (g306) was investigated to reveal the association between NRPS and nematocidal activity in the nematode-trapping fungus Arthrobotrys oligospora. Sequence analysis indicated that the encoded product of g306 is an adenylation domain of non-ribosomal peptide synthetases and extended short-chain dehydrogenase/reductase domain-containing proteins, and displays a wide substrate spectrum. The Δg306 mutants were more sensitive to chemical stressors than the wild type. Disruption of g306 impeded the nematocidal efficiency of A. oligospora. Metabolomics analysis showed that secondary metabolite biosynthesis and lipid metabolism were altered in the mutants. The phenotypic changes in the mutants can be attributed to the down-regulation of various metabolites, including fatty acyls, prenol lipids, steroidsand steroid derivative, and amino acid derivatives, identified in the present study. This study investigated the association between the non-ribosomal polypeptide-encoding gene g306 and nematicidal activity in A. oligospora, providing a reference for resolving the predation mechanism of nematode-trapping fungus.

AfLaeA, a Global Regulator of Mycelial Growth, Chlamydospore Production, Pathogenicity, Secondary Metabolism, and Energy Metabolism in the Nematode-Trapping Fungus Arthrobotrys flagrans.

Arthrobotrys flagrans (Duddingtonia flagrans) is a typical nematode-trapping fungus which has been used for nematode biocontrol. The global regulator LaeA is widely distributed in filamentous fungi and plays a crucial role in secondary metabolism and development in addition to pathogenicity in fungal pathogens. In this study, the chromosome-level genome of A. flagrans CBS 565.50 was sequenced and homologous sequences of LaeA were identified in A. flagrans. A. flagrans LaeA (AfLaeA) knockout resulted in slower hyphal growth and a smoother hyphal surface. Importantly, deletion of AfLaeA resulted in the absence of chlamydospores and attenuated glycogen and lipid accumulation in hyphae. Similarly, disruption of the AfLaeA gene led to fewer traps and electron-dense bodies, lower protease activity, and a delay in capturing nematodes. The AfLaeA gene had a large effect on the secondary metabolism of A. flagrans, and both the deletion and overexpression of AfLaeA could yield new compounds, whereas some compounds were lost due to the absence of the AfLaeA. Protein-protein interactions between AfLaeA and another eight proteins were detected. Furthermore, transcriptome data analysis showed that 17.77% and 35.51% of the genes were influenced by the AfLaeA gene on days 3 and 7, respectively. AfLaeA gene deletion resulted in the higher expression level of the artA gene cluster, and multiple differentially expressed genes involved in glycogen and lipid synthesis and metabolism showed opposite expression patterns in wild-type and ΔAfLaeA strains. In summary, our results provide novel insights into the functions of AfLaeA in mycelial growth, chlamydospore production, pathogenicity, secondary metabolism, and energy metabolism in A. flagrans. IMPORTANCE The regulation of biological functions, such as the secondary metabolism, development, and pathogenicity of LaeA, has been reported in multiple fungi. But to date, no study on LaeA in nematode-trapping fungi has been reported. Moreover, it has not been investigated whether or not LaeA is involved in energy metabolism and chlamydospore formation has not been investigated. Especially in the formation mechanism of chlamydospores, several transcription factors and signaling pathways are involved in the production of chlamydospores, but the mechanism of chlamydospore formation from an epigenetic perspective has not been revealed. Concurrently, an understanding of protein-protein interactions will provide a broader perspective on the regulatory mechanism of AfLaeA in A. flagrans. This finding is critical for understanding the regulatory role of AfLaeA in the biocontrol fungus A. flagrans and establishes a foundation for developing high-efficiency nematode biocontrol agents.

Tools and basic procedures of gene manipulation in nematode-trapping fungi.

Nematode-trapping fungi (NTF) are the majority of carnivorous microbes to capture nematodes through diverse and sophisticated trapping organs derived from hyphae. They can adopt carnivorous lifestyles in addition to saprophytism to obtain extra-nutrition from nematodes. As a special group of fungi, the NTF are not only excellent model organism for studying lifestyle transition of fungi but also natural resources of exploring biological control of nematodes. However, the carnivorous mechanism of NTF remains poorly understood. Nowadays, the omics studies of NTF have provided numerous genes and pathways that are associated with the phenotypes of carnivorous traits, which need molecular tools to verify. Here, we review the development and progress of gene manipulation tools in NTF, including methodology and strategy of transformation, random gene mutagenesis methods and target gene mutagenesis methods. The principle and practical approach for each method was summarized and discussed, and the basic operational flow for each tool was described. This paper offers a clear reference and instruction for researchers who work on NTF as well as other group of fungi.

Arthrobotrys blastospora sp. nov. (Orbiliomycetes): A Living Fossil Displaying Morphological Traits of Mesozoic Carnivorous Fungi.

The evolution of carnivorous fungi in deep time is still poorly understood as their fossil record is scarce. The approximately 100-million-year-old Cretaceous Palaeoanellus dimorphus is the earliest fossil of carnivorous fungi ever discovered. However, its accuracy and ancestral position has been widely questioned because no similar species have been found in modern ecosystems. During a survey of carnivorous fungi in Yunnan, China, two fungal isolates strongly morphologically resembling P. dimorphus were discovered and identified as a new species of Arthrobotrys (Orbiliaceae, Orbiliomycetes), a modern genus of carnivorous fungi. Phylogenetically, Arthrobotrys blastospora sp. nov. forms a sister lineage to A. oligospora. A. blastospora catches nematodes with adhesive networks and produces yeast-like blastospores. This character combination is absent in all other previously known modern carnivorous fungi but is strikingly similar to the Cretaceous P. dimorphus. In this paper, we describe A. blastospora in detail and discuss its relationship to P. dimorphus.

Characterization of the complete mitochondrial genome of the nematode-trapping fungus Drechslerella dactyloides.

The complete mitochondrial genome of Drechslerella dactyloides was characterized in this study. This mitogenome is a closed circular molecule of 246860 bp in length with a GC content of 26.16%, including 87 predicted protein-coding genes, 29 transfer RNA genes, and two rRNA gens. Phylogenetic analyses based on concatenated amino acid sequences at 14 conserved mitochondrial protein-coding genes showed that D. dactyloides was closely related to Dactylellina haptotyla.

Vermicomposting of Pleurotus eryngii spent mushroom substrates and the possible mechanisms of vermicompost suppressing nematode disease caused by Meloidogyne incognita.

The mushroom industry produces a large amount of spent mushroom substrate (SMS), which requires a large geographical footprint and causes pollution. Vermicomposting is a low-cost technology for its value in recycling of organic wastes and production of beneficial organic fertilizers. In this study, the changes of physicochemical properties was characterized during vermicomposting of Pleurotus eryngii SMS with cow dung (CD) as amendment. The efficiency and possible mechanisms of vermicompost suppressing disease induced by Meloidogyne incognita was also investigated. Six combinations with different ratios of SMS and cow dung (CD) was included in the vermicomposting using Eisenia fetida. Effect of vermicompost against disease induced by M. incognita on tobacco was conducted under greenhouse condition. And the possible mechanisms of vermicompost suppressing M. incognita was investigated by evaluated the species diversity of nematode-trapping fungi (NTF) in soil, and the defense response enzymes in tobacco. The combination of 65% SMS +35% CD was more suitable for vermicomposting, in which the highest vermicompost production (57%) and earthworm biomass increment (268%) were achieved. Additionally, the reduction in pH, total organic carbon, carbon: nitrogen ratio, and the pronounced elevation in four overall nutrient status were also observed. Soil amended with vermicompost (100:1 w/w) showed 61% control efficiency against nematode disease caused by M. incognita on tobacco, which significantly higher than that of the normal compost (24%). Comparing to the normal compost, the potential mechanism of vermicompost suppressing M. incognita could be rely on promoting species diversity of NTF in soil and enhancing the activities of the defense response enzymes in tobacco plant. Our findings indicate that vermicomposting is a promising technology for recycling of P. eryngii SMS, and the resulting vermicompost as organic fertilizer can be sued for management of the diseases caused by root-knot nematodes. This study establish a sustainable avenue for P. eryngii SMS disposal and a practical manner for controlling pathogens.

Recent Advances in Life History Transition with Nematode-Trapping Fungus Arthrobotrys oligospora and Its Application in Sustainable Agriculture.

Parasitic nematodes cause great annual loss in the agricultural industry globally. Arthrobotrys oligospora is the most prevalent and common nematode-trapping fungus (NTF) in the environment and the candidate for the control of plant- and animal-parasitic nematodes. A. oligospora is also the first recognized and intensively studied NTF species. This review highlights the recent research advances of A. oligospora as a model to study the biological signals of the switch from saprophytism to predation and their sophisticated mechanisms for interacting with their invertebrate hosts, which is of vital importance for improving the engineering of this species as an effective biocontrol fungus. The application of A. oligospora in industry and agriculture, especially as biological control agents for sustainable purposes, was summarized, and we discussed the increasing role of A. oligospora in studying its sexual morph and genetic transformation in complementing biological control research.

DhFIG_2, a gene of nematode-trapping fungus Dactylellina haptotyla that encodes a component of the low-affinity calcium uptake system, is required for conidiation and knob-trap formation.

Calcium ion (Ca2+) is a universal second messenger involved in regulating diverse processes in animals, plants, and fungi. The low-affinity calcium uptake system (LACS) participates in acquiring Ca2+ from extracellular environments under high extracellular Ca2+ concentration. Unlike most fungi, which encode only one protein (FIG1) for LACS, nematode-trapping fungi (NTF) encode two related proteins. AoFIG_2, the NTF-specific LACS component encoded by adhesive network-trap forming Arthrobotrys oligospora, was shown to be required for conidiation and trap formation. We characterized the role of DhFIG_2, an AoFIG_2 ortholog encoded by knob-trap forming Dactylellina haptotyla, in growth and development to expand our understanding of the role of LACS in NTF. Because repeated attempts to disrupt DhFIG_2 failed, knocking down the expression of DhFIG_2 via RNA interference (RNAi) was used to study its function. RNAi of DhFIG_2 significantly decreased its expression, severely reduced conidiation and trap formation, and affected vegetative growth and stress responses, suggesting that this component of LACS is crucial for trap formation and conidiation in NTF. Our study demonstrated the utility of RNAi assisted by ATMT for studying gene function in D. haptotyla.

The cAMP-PKA pathway regulates prey sensing and trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora.

Sensing environmental factors and responding swiftly to them is essential for all living organisms. For instance, predators must act rapidly once prey is sensed. Nematode-trapping fungi (NTF) are predators that use "traps" differentiated from vegetative hyphae to capture, kill, and consume nematodes. These traps undergo drastic and rapid morphological changes upon nematode induction. Multiple signaling hubs have been shown to regulate this remarkable process. Here, we demonstrate that the conserved cAMP-PKA signaling pathway exerts a crucial role in trap morphogenesis of the nematode-trapping fungi Arthrobotrys oligospora. A gene deletion mutant of the PKA catalytic subunit TPK2 proved insensitive toward nematode presence. Moreover, we show that the G protein alpha subunit GPA2 acts upstream of adenylate cyclase, with GPA2 deletion resulting in substantially reduced trap formation, whereas exogenous provision of cAMP rescued the prey-sensing and trap morphogenesis defects of a gpa2 mutant. Thus, we show that cAMP production triggered by G protein signaling and downstream PKA activity are vital for prey-sensing and trap development in A. oligospora, demonstrating that this highly conserved signaling pathway is critical for nematode-trapping fungi and nematode predator-prey interactions.

New Arthrobotrys Nematode-Trapping Species (Orbiliaceae) from Terrestrial Soils and Freshwater Sediments in China.

Arthrobotrys is the most complex genus of Orbiliaceae nematode-trapping fungi. Its members are widely distributed in various habitats worldwide due to their unique nematode-trapping survival strategies. During a survey of nematophagous fungi in Yunnan Province, China, twelve taxa were isolated from terrestrial soil and freshwater sediment habitats and were identified as six new species in Arthrobotrys based on evidence from morphological and multigene (ITS, TEF, and RPB2) phylogenetic analyses. These new species i.e., Arthrobotrys eryuanensis, A. jinpingensis, A. lanpingensis, A. luquanensis, A. shuifuensis, and A. zhaoyangensis are named in recognition of their places of origin. Morphological descriptions, illustrations, taxonomic notes, and a multilocus phylogenetic analysis are provided for all new taxa. In addition, a key to known species in Arthrobotrys is provided, and the inadequacies in the taxonomic study of nematode-trapping fungi are also discussed.