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.

Bacterial Volatile-Mediated Suppression of Root-Knot Nematode (Meloidogyne incognita).

Root-knot nematodes (Meloidogyne spp.) are obligate plant parasites that cause severe economic losses to agricultural crops worldwide. Because of serious health and environmental concerns related to the use of chemical nematicides, the development of efficient alternatives is of great importance. Biological control through exploiting the potential of rhizosphere microorganisms is currently accepted as an important approach for pest management in sustainable agriculture. In our research, during screening of rhizosphere bacteria against the root-knot nematodes Meloidogyne incognita, Ochrobactrum pseudogrignonense strain NC1 from the rhizosphere of healthy tomatoes showed strong nematode inhibition. A volatile nematicidal assay showed that the cell-free fermentation filtrate in the first-row wells of 12-well tissue culture plates caused M. incognita juvenile mortality in the second-row wells. Gas chromatography-mass spectrometry analysis revealed that dimethyl disulfide (DMDS) and benzaldehyde were the main volatile compounds produced by strain NC1. The nematicidal activity of these compounds indicated that the lethal concentration 50 against the M. incognita juveniles in the second-row wells and the fourth-row wells were 23.4 µmol/ml and 30.7 µmol/ml for DMDS and 4.7 µmol/ml and 15.2 µmol/ml for benzaldehyde, respectively. A greenhouse trial using O. pseudogrignonense strain NC1 provided management efficiencies of root-knot nematodes of 88 to 100% compared with the untreated control. This study demonstrated that nematode-induced root-gall suppression mediated by the bacterial volatiles DMDS and benzaldehyde presents a new opportunity for root-knot nematode management.

DdaSTE12 is involved in trap formation, ring inflation, conidiation, and vegetative growth in the nematode-trapping fungus Drechslerella dactyloides.

Ste12 transcription factors, downstream of mitogen-activated protein kinase (MAPK) signalling pathways, are exclusively found in the fungal kingdom and regulate fungal mating, development, and pathogenicity. The nematode-trapping fungus Drechslerella dactyloides can capture free-living nematodes using constricting rings by cell inflation within 1 s when stimulated by nematodes entering the rings. The MAPK signalling pathways are involved in the trap formation of nematode-trapping fungi, but their downstream regulation is not clearly understood. In this study, disruption of the DdaSTE12 gene in D. dactyloides disabled cell inflation of constricting rings and led to an inability to capture nematodes. The number of septa of constricting rings and the ring cell vacuoles were changed in ΔDdaSTE12. Compared with the wild type, ΔDdaSTE12 reduced trap formation, conidiation, and vegetative growth by 79.3%, 80.3%, and 21.5%, respectively. The transcriptomes of ΔDdaSTE12-3, compared with those of the wild type, indicated that the expression of genes participating in trap formation processes, including signal transduction (Gpa2 and a 7-transmembrane receptor), vesicular transport and cell fusion (MARVEL domain-containing proteins), and nematode infection (PEX11 and CFEM domain-containing proteins), is regulated by DdaSTE12. The results suggest that DdaSTE12 is involved in trap formation and ring cell inflation, as well as conidiation and vegetative growth, by regulating a wide range of downstream functions. Our findings expanded the roles of Ste12 homologous transcription factors in the development of constricting rings and provided new insights into the downstream regulation of the MAPK signalling pathway involved in nematode predation. KEY POINTS: • DdaSTE12 was the first gene disrupted in D. dactyloides. • DdaSTE12 is related to ring cell inflation, vegetative growth, and conidiation. • DdaSTE12 deletion resulted in defects in trap formation and ring development.

Anti-cryptococcal activity of preussolides A and B, phosphoethanolamine-substituted 24-membered macrolides, and leptosin C from coprophilous isolates of Preussia typharum.

Cryptococcus neoformans is a serious human pathogen with limited options for treatment. We have interrogated extracts from fungal fermentations to find Cryptococcus-inhibiting natural products using assays for growth inhibition and differential thermosensitivity. Extracts from fermentations of four fungal strains from wild and domestic animal dung from Arkansas and West Virginia, USA were identified as Preussia typharum. The extracts exhibited two antifungal regions. Purification of one region yielded new 24-carbon macrolides incorporating both a phosphoethanolamine unit and a bridging tetrahydrofuran ring. The structures of these metabolites were established mainly by analysis of high-resolution mass spectrometry and 2D NMR data. Relative configurations were assigned using NOESY data, and the structure assignments were supported by NMR comparison with similar compounds. These new metabolites are designated preussolides A and B. The second active region was caused by the cytotoxin, leptosin C. Genome sequencing of the four strains revealed biosynthetic gene clusters consistent with those known to encode phosphoethanolamine-bearing polyketide macrolides and the biosynthesis of dimeric epipolythiodioxopiperazines. All three compounds showed moderate to potent and selective antifungal activity toward the pathogenic yeast C. neoformans.

Population Genetics of Hirsutella rhossiliensis, a Dominant Parasite of Cyst Nematode Juveniles on a Continental Scale.

Hirsutella rhossiliensis is a parasite of juvenile nematodes, effective against a diversity of plant-parasitic nematodes. Its global distribution on various nematode hosts and its genetic variation for several geographic regions have been reported, while the global population genetic structure and factors underlying patterns of genetic variation of H. rhossiliensis are unclear. In this study, 87 H. rhossiliensis strains from five nematode species (Globodera sp., Criconemella xenoplax, Rotylenchus robustus, Heterodera schachtii, and Heterodera glycines) in Europe, the United States, and China were investigated by multilocus sequence analyses. A total of 280 variable sites (frequency, 0.6%) at eight loci and six clustering in high accordance with geographic populations or host nematode-associated populations were identified. Although H. rhossiliensis is currently recognized as an asexual fungus, recombination events were frequently detected. In addition, significant genetic isolation by geography and nematode hosts was revealed. Overall, our analyses showed that recombination, geographic isolation, and nematode host adaptation have played significant roles in the evolutionary history of H. rhossiliensis IMPORTANCE: H. rhossiliensis has great potential for use as a biocontrol agent to control nematodes in a sustainable manner as an endoparasitic fungus. Therefore, this study has important implications for the use of H. rhossiliensis as a biocontrol agent and provides interesting insights into the biology of this species.

Genomic and transcriptomic analysis of the endophytic fungus Pestalotiopsis fici reveals its lifestyle and high potential for synthesis of natural products.

BACKGROUND: In recent years, the genus Pestalotiopsis is receiving increasing attention, not only because of its economic impact as a plant pathogen but also as a commonly isolated endophyte which is an important source of bioactive natural products. Pestalotiopsis fici Steyaert W106-1/CGMCC3.15140 as an endophyte of tea produces numerous novel secondary metabolites, including chloropupukeananin, a derivative of chlorinated pupukeanane that is first discovered in fungi. Some of them might be important as the drug leads for future pharmaceutics. RESULTS: Here, we report the genome sequence of the endophytic fungus of tea Pestalotiopsis fici W106-1/CGMCC3.15140. The abundant carbohydrate-active enzymes especially significantly expanding pectinases allow the fungus to utilize the limited intercellular nutrients within the host plants, suggesting adaptation of the fungus to endophytic lifestyle. The P. fici genome encodes a rich set of secondary metabolite synthesis genes, including 27 polyketide synthases (PKSs), 12 non-ribosomal peptide synthases (NRPSs), five dimethylallyl tryptophan synthases, four putative PKS-like enzymes, 15 putative NRPS-like enzymes, 15 terpenoid synthases, seven terpenoid cyclases, seven fatty-acid synthases, and five hybrids of PKS-NRPS. The majority of these core enzymes distributed into 74 secondary metabolite clusters. The putative Diels-Alderase genes have undergone expansion. CONCLUSION: The significant expansion of pectinase encoding genes provides essential insight in the life strategy of endophytes, and richness of gene clusters for secondary metabolites reveals high potential of natural products of endophytic fungi.

pH dependency of sclerotial development and pathogenicity revealed by using genetically defined oxalate-minus mutants of Sclerotinia sclerotiorum.

The devastating plant pathogen Sclerotinia sclerotiorum produces copious (up to 50 mM) amounts of oxalic acid, which, for over a quarter century, has been claimed as the pathogenicity determinant based on UV-induced mutants that concomitantly lost oxalate production and pathogenicity. Such a claim was made without fulfilling the molecular Koch's postulates because the UV mutants are genetically undefined and harbour a developmental defect in sclerotial production. Here, we generated oxalate-minus mutants of S. sclerotiorum using two independent mutagenesis techniques, and tested the resulting mutants for growth at different pHs and for pathogenicity on four host plants. The oxalate-minus mutants accumulated fumaric acid, produced functional sclerotia and have reduced ability to acidify the environment. The oxalate-minus mutants retained pathogenicity on plants, but their virulence varied depending on the pH and buffering capacity of host tissue. Acidifying the host tissue enhanced virulence of the oxalate-minus mutants, whereas supplementing with oxalate did not. These results suggest that it is low pH, not oxalic acid itself, that establishes the optimum conditions for growth, reproduction, pathogenicity and virulence expression of S. sclerotiorum. Exonerating oxalic acid as the primary pathogenicity determinant will stimulate research into identifying additional candidates as pathogenicity factors towards better understanding and managing Sclerotinia diseases.

Genetic structure and parasitization-related ability divergence of a nematode fungal pathogen Hirsutella minnesotensis following founder effect in China.

The fungal parasitoid, Hirsutella minnesotensis, is a dominant parasitoid of the soybean cyst nematode, which is a destruction pest of soybean crops. We investigated population structure and parasitism pattern in samples of H. minnesotensis in China to reveal the spreading pattern of this fungal species and the underlying mechanism generating the parasitization-related ability variability in Chinese population. In cross-inoculation experiments using different combinations of H. minnesotensis and soybean cyst nematode samples from China, most H. minnesotensis isolates fitted the criterion for "local versus foreign" parasitism profile, exhibiting local adaptation pattern to the SCN host. However, the genetic analysis of the single nucleotide polymorphisms with clone-corrected samples based on ten DNA fragments in 56 isolates of H. minnesotensis from China revealed that the Chinese H. minnesotensis population was a clonal lineage that underwent a founder event. The results demonstrated that the Chinese H. minnesotensis population had generated parasitization-related ability diversity after a founder event through individual variation or phenotypic plasticity other than local adaptation. The rapid divergence of parasitization-related abilities with simple genetic structure in Chinese H. minnesotensis population indicates a fundamental potential for the establishment of invasive fungal species, which is a prerequisite for biological control agents.

Engineering of Glarea lozoyensis for exclusive production of the pneumocandin B0 precursor of the antifungal drug caspofungin acetate.

Pneumocandins produced by the fungus Glarea lozoyensis are acylated cyclic hexapeptides of the echinocandin family. Pneumocandin B0 is the starting molecule for the first semisynthetic echinocandin antifungal drug, caspofungin acetate. In the wild-type strain, pneumocandin B0 is a minor fermentation product, and its industrial production was achieved by a combination of extensive mutation and medium optimization. The pneumocandin biosynthetic gene cluster was previously elucidated by a whole-genome sequencing approach. Knowledge of the biosynthetic cluster suggested an alternative way to produce exclusively pneumocandin B0. Disruption of GLOXY4, encoding a nonheme, α-ketoglutarate-dependent oxygenase, confirmed its involvement in l-leucine cyclization to form 4S-methyl-l-proline. The absence of 4S-methyl-l-proline abolishes pneumocandin A0 production, and 3S-hydroxyl-l-proline occupies the hexapeptide core's position 6, resulting in exclusive production of pneumocandin B0. Retrospective analysis of the GLOXY4 gene in a previously isolated pneumocandin B0-exclusive mutant (ATCC 74030) indicated that chemical mutagenesis disrupted the GLOXY4 gene function by introducing two amino acid mutations in GLOXY4. This one-step genetic manipulation can rationally engineer a high-yield production strain.

Origin and evolution of carnivorism in the Ascomycota (fungi).

Carnivorism is one of the basic life strategies of fungi. Carnivorous fungi possess the ability to trap and digest their preys by sophisticated trapping devices. However, the origin and development of fungal carnivorism remains a gap in evolution biology. In this study, five protein-encoding genes were used to construct the phylogeny of the carnivorous fungi in the phylum Ascomycota; these fungi prey on nematodes by means of specialized trapping structures such as constricting rings and adhesive traps. Our analysis revealed a definitive pattern of evolutionary development for these trapping structures. Molecular clock calibration based on two fossil records revealed that fungal carnivorism diverged from saprophytism about 419 Mya, which was after the origin of nematodes about 550-600 Mya. Active carnivorism (fungi with constricting rings) and passive carnivorism (fungi with adhesive traps) diverged from each other around 246 Mya, shortly after the occurrence of the Permian-Triassic extinction event about 251.4 Mya. The major adhesive traps evolved around 198-208 Mya, which was within the time frame of the Triassic-Jurassic extinction event about 201.4 Mya. However, no major carnivorous ascomycetes divergence was correlated to the Cretaceous-Tertiary extinction event, which occurred more recently (about 65.5 Mya). Therefore, a causal relationship between mass extinction events and fungal carnivorism evolution is not validated in this study. More evidence including additional fossil records is needed to establish if fungal carnivorism evolution was a response to mass extinction events.

Rupestriomyces and Spissiomyces, two new genera of rock-inhabiting fungi from China.

Bare rocks in arid and semi-arid climates may harbor a bewildering biodiversity of fungi that are overlooked in China. During a survey of rock-inhibiting fungi in China, more than 1000 were isolated and 14 belonging to Dothideomycetes (Ascomycota) were selected for detailed study. Phylogenetic trees based on combined sequence datasets of mt 16S rDNA, partial nuc rDNA 18S and 28S indicated that these strains clustered in two distinct, well supported and previously unknown lineages within the class Dothideomycetes. Therefore two new genera were established corresponding to those two clades. Spissiomyces gen. nov. is characterized by thick-walled, yellowish brown hyphae and globose or subglobose conidia, if present. Rupestriomyces gen. nov. is characterized by globose, barrel-shaped, ampulliform or ovoidal conidia formed from acropetal, catenate hyphae. Further phylogenetic analyses using combined sequence datasets of the rDNA internal transcribed spacer (ITS) region and 16S rDNA, part of genes of RPB2, TUB2, nuc rDNA 18S and 28S revealed that those strains represented five new species (i.e. Spissiomyces aggregatus, S. ramosus, Rupestriomyces sinensis, R. ampulliformis, R. torulosus). They were described, illustrated and compared with similar taxa based on morphological characteristics and phylogenetic relationships. The results of this paper provides insight into the richness and diversity of rock-inhibiting fungi in nature.

Drechslerella stenobrocha genome illustrates the mechanism of constricting rings and the origin of nematode predation in fungi.

BACKGROUND: Nematode-trapping fungi are a unique group of organisms that can capture nematodes using sophisticated trapping structures. The genome of Drechslerella stenobrocha, a constricting-ring-forming fungus, has been sequenced and reported, and provided new insights into the evolutionary origins of nematode predation in fungi, the trapping mechanisms, and the dual lifestyles of saprophagy and predation. RESULTS: The genome of the fungus Drechslerella stenobrocha, which mechanically traps nematodes using a constricting ring, was sequenced. The genome was 29.02 Mb in size and was found rare instances of transposons and repeat induced point mutations, than that of Arthrobotrys oligospora. The functional proteins involved in nematode-infection, such as chitinases, subtilisins, and adhesive proteins, underwent a significant expansion in the A. oligospora genome, while there were fewer lectin genes that mediate fungus-nematode recognition in the D. stenobrocha genome. The carbohydrate-degrading enzyme catalogs in both species were similar to those of efficient cellulolytic fungi, suggesting a saprophytic origin of nematode-trapping fungi. In D. stenobrocha, the down-regulation of saprophytic enzyme genes and the up-regulation of infection-related genes during the capture of nematodes indicated a transition between dual life strategies of saprophagy and predation. The transcriptional profiles also indicated that trap formation was related to the protein kinase C (PKC) signal pathway and regulated by Zn(2)-C6 type transcription factors. CONCLUSIONS: The genome of D. stenobrocha provides support for the hypothesis that nematode trapping fungi evolved from saprophytic fungi in a high carbon and low nitrogen environment. It reveals the transition between saprophagy and predation of these fungi and also proves new insights into the mechanisms of mechanical trapping.

Phylogeography and evolution of a fungal-insect association on the Tibetan Plateau.

Parasitoidism refers to a major form of interspecies interactions where parasitoids sterilize and/or kill their hosts typically before hosts reach reproductive age. However, relatively little is known about the evolutionary dynamics of parasitoidism. Here, we investigate the spatial patterns of genetic variation of Chinese cordyceps, including both the parasitoidal fungus Ophiocordyceps sinensis and its host insects. We sampled broadly from alpine regions on the Tibetan Plateau and obtained sequences on seven fungal and three insect DNA fragments from each of the 125 samples. Seven and five divergent lineages/cryptic species were identified within the fungus and host insects, respectively. Our analyses suggested that O. sinensis and host insects originated at similar geographic regions in southern Tibet/Yunnan, followed by range expansion to their current distributions. Cophylogenetic analyses revealed a complex evolutionary relationship between O. sinensis and its host insects. Significant congruence was found between host and parasite phylogenies and the time estimates of divergence were similar, raising the possibility of the occurrence of cospeciation events, but the incongruences suggested that host shifts were also prevalent. Interestingly, one fungal genotype was broadly distributed, consistent with recent gene flow. In contrast, the high-frequency insect genotypes showed limited geographic distributions. The dominant genotypes from both the fungus and the insect hosts may represent ideal materials from which to develop artificial cultivation of this important Chinese traditional medicine. Our results demonstrate that both historical and contemporary events have played important roles in the phylogeography and evolution of the O. sinensis-ghost moth parasitoidism on the Tibetan Plateau.

A novel high-throughput nematicidal assay using embryo cells and larvae of Caenorhabditis elegans.

Human health safety and environmental concerns have resulted in the widespread deregistration of several agronomic important nematicides. New and safer nematicides are urgently needed. However, a high-throughput bioassay for screening potential nematicides has not been established. We developed a two-step high-throughput nematicidal screening method to combine a cell-based MTS colorimetric assay with Caenorhabditis elegans embryo cells for preliminary cytotoxicity screening (step 1) followed by in vitro larval assay for nematicidal activity (step 2). Based on three conventional nematicides' test, high correlations were obtained between cell viability and larval viability and "r" values were 0.78 for Avermectin, 0.95 for Fosthiazate, and 0.65 for Formaldehyde solution. Further assays with 60 fungal secondary metabolites (extracts, fractions and pure compounds) also demonstrated the high correlation between cell viability and larval viability (r=0.60) and between the C. elegans cell viability and the juvenile viability of soybean cyst nematode Heterodera glycines (r=0.48) and pine wood nematode Bursaphelenchus xylophilus (r=0.56). Six metabolites with high cytotoxicity have performed high larval mortality with a LC50 range of 6.8-500µg/ml. These results indicate that the proposed two-step screening assay represents an efficient and labor-saving method for screening natural nematicidal products.

Wild plant species with broader precipitation niches exhibit stronger host selection in rhizosphere microbiome assembly.

Plants actively recruit microbes from the soil, forming species-specific root microbiomes. However, their relationship with plant adaptations to temperature and precipitation remains unclear. Here we examined the host-selected and conserved microbiomes of 13 native plant species in the Xilingol steppe, Inner Mongolia, a semi-arid region in China. By calculating the global precipitation and temperature niches of these plants, considering plant phylogenetic distances, and analyzing functional traits, we found that these factors significantly influenced the rhizosphere microbiome assembly. We further quantified the strength of host selection and observed that plants with wider precipitation niches exhibited greater host selection strength in their rhizosphere microbiome assembly and higher rhizosphere bacterial diversity. In general, the rhizosphere microbiome showed a stronger link to plant precipitation niches than temperature niches. Haliangium exhibited consistent responsiveness to host characteristics. Our findings offer novel insights into host selection effects and the ecological determinants of wild plant rhizosphere microbiome assembly, with implications for steering root microbiomes of wild plants and understanding plant-microbiome 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.

Microbiome-mediated alleviation of tobacco replant problem via autotoxin degradation after long-term continuous cropping.

Continuous cropping often results in severe "replant problem," across various crops due to the autotoxins accumulation, soil acidification, pathogens proliferation, and microbial dysfunction. We unveiled a groundbreaking phenomenon that long-term continuous cropping (LTCC) can alleviate the tobacco replant problem. This mitigation occurs through the enrichment of autotoxin-degrading microbes, and the transformative impact is evident with even a modest application (10%) of LTCC soil to short-term continuous cropping (STCC) soil. Our investigation has pinpointed specific autotoxin-degrading bacteria, particularly the Pseudomonas and Burkholderia species, which exhibit the capacity to alleviate the tobacco replant problem in STCC soil. Their autotoxin-degrading mechanism using axenic culture and soil samples was also conducted via comprehensive analyses of microbiome and transcriptome approach. This research sheds light on the potential of LTCC as a strategic approach for sustainable agriculture, addressing replant problems and promoting the health of cropping systems. UV, ultraviolet; OD, optical density.

Redundancy in microbiota-mediated suppression of the soybean cyst nematode.

BACKGROUND: Soybean cyst nematodes (SCN) as animal parasites of plants are not usually interested in killing the host but are rather focused on completing their life cycle to increase population, resulting in substantial yield losses. Remarkably, some agricultural soils after long-term crop monoculture show a significant decline in SCN densities and suppress disease in a sustainable and viable manner. However, relatively little is known about the microbes and mechanisms operating against SCN in such disease-suppressive soils. RESULTS: Greenhouse experiments showed that suppressive soils (S) collected from two provinces of China and transplantation soils (CS, created by mixing 10% S with 90% conducive soils) suppressed SCN. However, SCN suppressiveness was partially lost or completely abolished when S soils were treated with heat (80 °C) and formalin. Bacterial community analysis revealed that the specific suppression in S and CS was mainly associated with the bacterial phylum Bacteroidetes, specifically due to the enrichment of Chitinophaga spp. and Dyadobacter sp., in the cysts. SCN cysts colonized by Chitinophaga spp. showed dramatically reduced egg hatching, with unrecognizable internal body organization of juveniles inside the eggshell due to chitinase activity. Whereas, Dyadobacter sp. cells attached to the surface coat of J2s increased soybean resistance against SCN by triggering the expression of defence-associated genes. The disease-suppressive potential of these bacteria was validated by inoculating them into conducive soil. The Dyadobacter strain alone or in combination with Chitinophaga strains significantly decreased egg densities after one growing cycle of soybeans. In contrast, Chitinophaga strains alone required more than one growing cycle to significantly reduce SCN egg hatching and population density. CONCLUSION: This study revealed how soybean monoculture for decades induced microbiota homeostasis, leading to the formation of SCN-suppressive soil. The high relative abundance of antagonistic bacteria in the cyst suppressed the SCN population both directly and indirectly. Because uncontrolled proliferation will likely lead to quick demise due to host population collapse, obligate parasites like SCN may have evolved to modulate virulence/proliferation to balance these conflicting needs. Video Abstract.

Genomics-driven discovery of the pneumocandin biosynthetic gene cluster in the fungus Glarea lozoyensis.

BACKGROUND: The antifungal therapy caspofungin is a semi-synthetic derivative of pneumocandin B0, a lipohexapeptide produced by the fungus Glarea lozoyensis, and was the first member of the echinocandin class approved for human therapy. The nonribosomal peptide synthetase (NRPS)-polyketide synthases (PKS) gene cluster responsible for pneumocandin biosynthesis from G. lozoyensis has not been elucidated to date. In this study, we report the elucidation of the pneumocandin biosynthetic gene cluster by whole genome sequencing of the G. lozoyensis wild-type strain ATCC 20868. RESULTS: The pneumocandin biosynthetic gene cluster contains a NRPS (GLNRPS4) and a PKS (GLPKS4) arranged in tandem, two cytochrome P450 monooxygenases, seven other modifying enzymes, and genes for L-homotyrosine biosynthesis, a component of the peptide core. Thus, the pneumocandin biosynthetic gene cluster is significantly more autonomous and organized than that of the recently characterized echinocandin B gene cluster. Disruption mutants of GLNRPS4 and GLPKS4 no longer produced the pneumocandins (A0 and B0), and the Δglnrps4 and Δglpks4 mutants lost antifungal activity against the human pathogenic fungus Candida albicans. In addition to pneumocandins, the G. lozoyensis genome encodes a rich repertoire of natural product-encoding genes including 24 PKSs, six NRPSs, five PKS-NRPS hybrids, two dimethylallyl tryptophan synthases, and 14 terpene synthases. CONCLUSIONS: Characterization of the gene cluster provides a blueprint for engineering new pneumocandin derivatives with improved pharmacological properties. Whole genome estimation of the secondary metabolite-encoding genes from G. lozoyensis provides yet another example of the huge potential for drug discovery from natural products from the fungal kingdom.

Parasitism of secondary-stage juvenile of Heterodera glycines and four larva stages of Caenorhabditis elegans by Hirsutella spp.

The fungi Hirsutella rhossiliensis and Hirsutella minnesotensis generally parasitize only plant-parasitic nematodes in nature but parasitize the bacterivorous nematode Caenorhabditis elegans on agar plates. To establish a model system for studying the interaction between fungi and nematodes, we compared the parasitism of the first- to fourth-stage larvae (L1-L4) of C. elegans and second-stage juvenile (J2) of Heterodera glycines by twenty isolates of Hirsutella spp. Although parasitism differed substantially among isolates, both H. minnesotensis and H. rhossiliensis parasitized a higher percentage of H. glycines J2s than of C. elegans larvae. Parasitism of C. elegans L1s was correlated with parasitism of H. glycines J2s. Parasitism of C. elegans by H. rhossiliensis and H. minnesotensis was negatively correlated with larva size and motility, i.e., parasitism was higher for the younger stages. The C. elegans L1 is recommended for studying parasitism of nematodes by H. rhossiliensis and H. minnesotensis.