Emerald Ash Borer Management and Research: Decades of Damage and Still Expanding.

Since the discovery of the ash tree (Fraxinus spp.) killer emerald ash borer (EAB; Agrilus planipennis) in the United States in 2002 and Moscow, Russia in 2003, substantial detection and management efforts have been applied to contain and monitor its spread and mitigate impacts. Despite these efforts, the pest continues to spread within North America. It has spread to European Russia and Ukraine and is causing sporadic outbreaks in its native range in China. The dynamics of EAB's range expansion events appear to be linked to the lack of resistant ash trees in invaded ranges, facilitated by the abundance of native or planted North American susceptible ash species. We review recently gained knowledge of the range expansion of EAB; its ecological, economic, and social impacts; and past management efforts with their successes and limitations. We also highlight advances in biological control, mechanisms of ash resistance, and new detection and management approaches under development, with the aim of guiding more effective management.

Distinct metabolites affect the phloem fungal communities in ash trees (Fraxinus spp.) native and nonnative to the highly invasive emerald ash borer (AGRILUS PLANIPENNIS).

Emerald ash borer (EAB, Agrilus planipennis) is an invasive killer of ash trees (Fraxinus spp.) in North America and Europe. Ash species co-evolved with EAB in their native range in Asia are mostly resistant, although the precise mechanism(s) remain unclear. Very little is also known about EAB or ash tree microbiomes. We performed the first joint comparison of phloem mycobiome and metabolites between a native and a nonnative ash species, infested and uninfested with EAB, in conjunction with investigation of larval mycobiome. Phloem mycobiome communities differed between the tree species, but both were unaffected by EAB infestation. Several indicator taxa in the larval gut shared a similarly high relative abundance only with the native host trees. Widely targeted metabolomics revealed 24 distinct metabolites in native trees and 53 metabolites in nonnative trees, respectively, that differed in relative content between infested and uninfested trees only in one species. Interestingly, four metabolites shared a strong relationship with the phloem mycobiomes, majority of which affected only the native trees. Collectively, our results demonstrate a complex interplay between host tree chemistry and mycobiome, and suggest the shared relationships between the mycobiomes of the native host tree and EAB may reflect their shared co-evolution.

Sources of Fungal Symbionts in the Microbiome of a Mobile Insect Host, Spodoptera frugiperda.

The sources of fungal symbionts of insects are not well understood, yet the acquisition and assembly of fungal communities in mobile insect hosts have important implications for the ecology of migratory insects and their plant hosts. To determine potential sources of fungi associated with the fall armyworm (Spodoptera frugiperda), we characterized the fungal communities associated with four different ecological compartments (insects, infested leaves, uninfested leaves, and soil) and estimated the contributions of each of these potential sources to the insect's fungal microbiome. Results show that insect fungal community composition was distinct from and more varied than the composition of fungal communities in the environment of those insects (plants and soil). Among the sources evaluated, on average we found a surprisingly large apparent contribution from other congeneric S. frugiperda insect larvae (ca. 25%) compared to the contribution from soil or plant sources (< 5%). However, a large proportion of the insect microbiome could not be attributed to the sampled sources and was instead attributed to unknown sources (ca. 50%). Surprisingly, we found little evidence for exchange of fungal taxa, with the exception of a Fusarium oxysporum and a Cladosporium sp. OTU, between larvae and the infested leaves on which they fed. Together, our results suggest that mobile insects such as S. frugiperda obtain their fungal symbionts from a variety of sources, not limited to plants and soil, but including conspecific insects and other unsampled environmental sources, and that transmission among insects may play an important role in acquisition of fungal symbionts.

Gene family expansion of pinewood nematode to detoxify its host defence chemicals.

Gene gain/loss in the context of gene family dynamics plays an important role in evolutionary processes as organisms, particularly invasive species, adapt to new environments or niches. One notable example of this is the duplication of digestive proteases in some parasitic insects and helminths to meet nutritional requirements during animal parasitism. However, whether gene family expansion participates in the adaptation of a plant parasite nematode to its host remains unknown. Here, we compared the newly sequenced genomes of the pinewood nematode, Bursaphelenchus xylophilus, with the genomes of free-living, animal-parasitic and plant-parasitic nematodes. The results showed gene expansions occurring in 51 gene families in B. xylophilus, especially in xenobiotic detoxification pathways, including flavin monooxygenase (FMO), cytochrome P450 (CYP450), short chain dehydrogenase (SDR), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST). Although a majority of these expansions probably resulted from gene duplications, nine ADH genes were potentially acquired by horizontal gene transfer (HGT) from fungi. From the transcriptomes of B. xylophilus treated with pine saplings and terpenes, candidate xenobiotic detoxification genes were identified. We propose that host defence chemicals led to gene family expansions of xenobiotic detoxification pathways in B. xylophilus facilitating its survival in pine resin ducts. This study contributes to a better understanding of how a parasitic nematode adapts to its host.

In Vitro Screening of a Culturable Soybean Cyst Nematode Cyst Mycobiome for Potential Biological Control Agents and Biopesticides.

Fungal biological control of soybean cyst nematodes (SCN) is an important component of integrated pest management for soybean. However, very few fungal biological control agents are available in the market. In this study, we have screened fungi previously isolated from SCN cysts over 3 years from a long-term crop rotation field experiment for their ability to antagonize SCN using (i) parasitism, (ii) egg hatch inhibition, and (iii) J2 mortality. We evaluated egg parasitism using an in-vitro egg parasitism bioassays and scored parasitism using the egg parasitic index (EPI) and fluorescent microscopy. The ability of these fungi to produce metabolites causing egg hatch inhibition and J2 mortality was assessed in bioassays using filter-sterilized culture filtrates. We identified 10 high-performing isolates each for egg parasitism and toxicity toward SCN eggs and J2s and repeated the tests after storage for 1 year of cryopreservation at -80°C to validate the durability of biocontrol potential of the chosen 20 isolates. Although the parasitic ability changed slightly for the majority of strains after cryopreservation, they still scored 5/10 on EPI scales. There were no differences in the ability of fungi to produce antinemic metabolites after cryopreservation.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Fungal communities associated with Heterodera glycines and their potential in biological control: a current update.

The soybean cyst nematode (SCN) is the most important pest on soybean, a major crop worldwide. The SCN is considered both parasitic and pathogenic as it derives nutrition from the host and manipulates host physiology to do so. Currently, there are no commercially available chemicals that are specific, environmentally safe and cost effective to control SCN levels. Crop rotation, use of host resistance and other cultural practices remain the main management strategies. The need for bioprospecting other methods of controlling SCN is paramount, and fungi show promise in that respect. Several studies have evaluated fungi and fungal products as biocontrol options against plant-parasitic nematodes. This review discusses fungal genera isolated from the SCN with potential for use as biocontrol agents and the effects of their secondary metabolites on various stages of SCN development. The review also summarizes efforts to control SCN using soil amendments that could potentially impact fungal communities in the soil.The soybean cyst nematode (SCN) is the most important pest on soybean, a major crop worldwide. The SCN is considered both parasitic and pathogenic as it derives nutrition from the host and manipulates host physiology to do so. Currently, there are no commercially available chemicals that are specific, environmentally safe and cost effective to control SCN levels. Crop rotation, use of host resistance and other cultural practices remain the main management strategies. The need for bioprospecting other methods of controlling SCN is paramount, and fungi show promise in that respect. Several studies have evaluated fungi and fungal products as biocontrol options against plant-parasitic nematodes. This review discusses fungal genera isolated from the SCN with potential for use as biocontrol agents and the effects of their secondary metabolites on various stages of SCN development. The review also summarizes efforts to control SCN using soil amendments that could potentially impact fungal communities in the soil.

Chromosome rearrangements shape the diversification of secondary metabolism in the cyclosporin producing fungus Tolypocladium inflatum.

BACKGROUND: Genes involved in production of secondary metabolites (SMs) in fungi are exceptionally diverse. Even strains of the same species may exhibit differences in metabolite production, a finding that has important implications for drug discovery. Unlike in other eukaryotes, genes producing SMs are often clustered and co-expressed in fungal genomes, but the genetic mechanisms involved in the creation and maintenance of these secondary metabolite biosynthetic gene clusters (SMBGCs) remains poorly understood. RESULTS: In order to address the role of genome architecture and chromosome scale structural variation in generating diversity of SMBGCs, we generated chromosome scale assemblies of six geographically diverse isolates of the insect pathogenic fungus Tolypocladium inflatum, producer of the multi-billion dollar lifesaving immunosuppressant drug cyclosporin, and utilized a Hi-C chromosome conformation capture approach to address the role of genome architecture and structural variation in generating intraspecific diversity in SMBGCs. Our results demonstrate that the exchange of DNA between heterologous chromosomes plays an important role in generating novelty in SMBGCs in fungi. In particular, we demonstrate movement of a polyketide synthase (PKS) and several adjacent genes by translocation to a new chromosome and genomic context, potentially generating a novel PKS cluster. We also provide evidence for inter-chromosomal recombination between nonribosomal peptide synthetases located within subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. In contrast, the cyclosporin cluster, located internally on chromosomes, was conserved across strains, suggesting selective maintenance of this important virulence factor for infection of insects. CONCLUSIONS: This research places the evolution of SMBGCs within the context of whole genome evolution and suggests a role for recombination between chromosomes in generating novel SMBGCs in the medicinal fungus Tolypocladium inflatum.

The genome of tolypocladium inflatum: evolution, organization, and expression of the cyclosporin biosynthetic gene cluster.

The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (simA) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology.

Comparative genome structure, secondary metabolite, and effector coding capacity across Cochliobolus pathogens.

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.

The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of antifungal peptaibiotics.

BACKGROUND: Two major mycoparasitic lineages, the family Hypocreaceae and the genus Tolypocladium, exist within the fungal order, Hypocreales. Peptaibiotics are a group of secondary metabolites almost exclusively described from Trichoderma species of Hypocreaceae. Peptaibiotics are produced by nonribosomal peptide synthetases (NRPSs) and have antibiotic and antifungal activities. Tolypocladium species are mainly truffle parasites, but a few species are insect pathogens. RESULTS: The draft genome sequence of the truffle parasite Tolypocladium ophioglossoides was generated and numerous secondary metabolite clusters were discovered, many of which have no known putative product. However, three large peptaibiotic gene clusters were identified using phylogenetic analyses. Peptaibiotic genes are absent from the predominantly plant and insect pathogenic lineages of Hypocreales, and are therefore exclusive to the largely mycoparasitic lineages. Using NRPS adenylation domain phylogenies and reconciliation of the domain tree with the organismal phylogeny, it is demonstrated that the distribution of these domains is likely not the product of horizontal gene transfer between mycoparasitic lineages, but represents independent losses in insect pathogenic lineages. Peptaibiotic genes are less conserved between species of Tolypocladium and are the product of complex patterns of lineage sorting and module duplication. In contrast, these genes are more conserved within the genus Trichoderma and consistent with diversification through speciation. CONCLUSIONS: Peptaibiotic NRPS genes are restricted to mycoparasitic lineages of Hypocreales, based on current sampling. Phylogenomics and comparative genomics can provide insights into the evolution of secondary metabolite genes, their distribution across a broader range of taxa, and their possible function related to host specificity.

Potential for Use of Species in the Subfamily Erynioideae for Biological Control and Biotechnology.

The fungal order Entomophthorales in the Zoopagomycota includes many fungal pathogens of arthropods. This review explores six genera in the subfamily Erynioideae within the family Entomophthoraceae, namely, Erynia, Furia, Orthomyces, Pandora, Strongwellsea, and Zoophthora. This is the largest subfamily in the Entomophthorales, including 126 described species. The species diversity, global distribution, and host range of this subfamily are summarized. Relatively few taxa are geographically widespread, and few have broad host ranges, which contrasts with many species with single reports from one location and one host species. The insect orders infected by the greatest numbers of species are the Diptera and Hemiptera. Across the subfamily, relatively few species have been cultivated in vitro, and those that have require more specialized media than many other fungi. Given their potential to attack arthropods and their position in the fungal evolutionary tree, we discuss which species might be adopted for biological control purposes or biotechnological innovations. Current challenges in the implementation of these species in biotechnology include the limited ability or difficulty in culturing many in vitro, a correlated paucity of genomic resources, and considerations regarding the host ranges of different species.

Fungi associated with galleries of the emerald ash borer.

The emerald ash borer (EAB) is an exotic forest pest that has killed millions of ash trees in the United States and Canada, resulting in an ecological disaster and billions of dollars in economic losses of urban landscape and forest trees. The beetle was first detected in Michigan in 2002 and has spread through much of the Eastern and Midwestern U.S., reaching Minnesota in 2009. Since then, it has spread across the state and poses a great risk to the more than 1 billion ash trees in Minnesota. The larval stage of EAB creates wounds on trees as they feed on the inner bark, causing disruption of water and sap flow that results in tree death. The fungal community associated with EAB larval galleries is poorly understood and the role these fungi may play in tree death is not known. This study describes fungi isolated from EAB larval galleries sampled throughout the main geographic areas of Minnesota where ash is affected by EAB. Fungal cultures were identified by extracting genomic DNA and sequencing the ITS region of the rDNA. Results from 1126 isolates reveal a diverse assemblage of fungi and three functional guilds comprised of canker pathogens, wood decay, and entomopathogenic fungi. The most common canker-associated genera were Cytospora followed by Phaeoacremonium, Paraconiothyrium, Coniothyrium, Nectria, Diplodia, and Botryosphaeria. Fungi in the Basidiomycota were nearly all wood decay causing fungi and many were species of pioneer colonizing genera including Sistotrema, Irpex, Peniophora, Phlebia and Ganoderma. Some of these fungi seriously affect urban trees, having the potential to cause rapid wood decay resulting in hazardous tree situations. Several entomopathogenic genera with the potential for biological control of EAB were also isolated from galleries. Purpureocillium was the most commonly isolated genus, followed by Beauveria, Clonostachys, Lecanicillium, Akanthomyces, Cordyceps, Microcera, Tolypocladium, and Pochonia. The results identify important fungal functional guilds that are occupying a new niche in ash trees resulting from EAB and include fungi that may accelerate decline in tree health, increase hazard tree situations, or may provide options for biological control of this destructive invasive insect.

Phylogenomics reveals subfamilies of fungal nonribosomal peptide synthetases and their evolutionary relationships.

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (NPS) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including alpha-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi. RESULTS: Phylogenomic analysis identified nine major subfamilies of fungal NRPSs which fell into two main groups: one corresponds to NPS genes encoding primarily mono/bi-modular enzymes which grouped with bacterial NRPSs and the other includes genes encoding primarily multimodular and exclusively fungal NRPSs. AARs shared a closer phylogenetic relationship to NRPSs than to other acyl-adenylating enzymes. Phylogenetic analyses and taxonomic distribution suggest that several mono/bi-modular subfamilies arose either prior to, or early in, the evolution of fungi, while two multimodular groups appear restricted to and expanded in fungi. The older mono/bi-modular subfamilies show conserved domain architectures suggestive of functional conservation, while multimodular NRPSs, particularly those unique to euascomycetes, show a diversity of architectures and of genetic mechanisms generating this diversity. CONCLUSIONS: This work is the first to characterize subfamilies of fungal NRPSs. Our analyses suggest that mono/bi-modular NRPSs have more ancient origins and more conserved domain architectures than most multimodular NRPSs. It also demonstrates that the alpha-aminoadipate reductases involved in lysine biosynthesis in fungi are closely related to mono/bi-modular NRPSs. Several groups of mono/bi-modular NRPS metabolites are predicted to play more pivotal roles in cellular metabolism than products of multimodular NRPSs. In contrast, multimodular subfamilies of NRPSs are of more recent origin, are restricted to fungi, show less stable domain architectures, and biosynthesize metabolites which perform more niche-specific functions than mono/bi-modular NRPS products. The euascomycete-only NRPS subfamily, in particular, shows evidence for extensive gain and loss of domains suggestive of the contribution of domain duplication and loss in responding to niche-specific pressures.

Natural variation of root lesion nematode antagonism in the biocontrol fungus Clonostachys rosea and identification of biocontrol factors through genome-wide association mapping.

Biological control is a promising approach to reduce plant diseases caused by nematodes to ensure high productivity in agricultural production. Large-scale analyses of genetic variation in fungal species used for biocontrol can generate knowledge regarding interaction mechanisms that can improve efficacy of biocontrol applications. In this study, we performed a genome-wide association study (GWAS) for in vitro antagonism against the root lesion nematode Pratylenchus penetrans in 53 previously genome re-sequenced strains of the biocontrol fungus Clonostachys rosea. Nematode mortality in C. rosea potato dextrose broth (PDB) culture filtrates was highly variable and showed continuous variation (p < .001) between strains, indicating a polygenic inheritance. Twenty-one strains produced culture filtrates with higher (p ≤ .05) nematode mortality compared with the PDB control treatment, while ten strains lowered (p ≤ .05) the mortality. The difference in in vitro antagonism against P. penetrans correlated with antagonism against the soybean cyst nematode Heterodera glycines, indicating lack of host specificity in C. rosea. An empirical Bayesian multiple hypothesis testing approach identified 279 single nucleotide polymorphism markers significantly (local false sign rate < 10-10) associated with the trait. Genes present in the genomic regions associated with nematicidal activity included several membrane transporters, a chitinase and genes encoding proteins predicted to biosynthesize secondary metabolites. Gene deletion strains of the predicted nonribosomal peptide synthetase genes nps4 and nps5 were generated and showed increased (p ≤ .001) fungal growth and conidiation rates compared to the wild type. Deletion strains also exhibited reduced (p < .001) nematicidal activity and reduced (p ≤ .05) biocontrol efficacy against nematode root disease and against fusarium foot rot on wheat. In summary, we show that the GWAS approach can be used to identify biocontrol factors in C. rosea, specifically the putative nonribosomal peptide synthetases NPS4 and NPS5.

RIP mutated ITS genes in populations of Ophiocordyceps sinensis and their implications for molecular systematics.

Different hypotheses have been proposed to interpret the observed unusual ITS (internal transcribed spacer) sequences in Ophiocordyceps sinensis. The coexistence of diverged ITS paralogs in a single genome was previously shown by amplifying the ITS region from mono-ascospore isolates using specific primers designed for different ITS paralog groups. Among those paralogs, are AT-biased ITS sequences which were hypothesized to result from repeat-induced point mutation (RIP). This is a process that detects and mutates repetitive DNA and frequently leads to epigenetic silencing, and these mutations have been interpreted as pseudogenes. Here we investigate the occurrence and frequency of ITS pseudogenes in populations of O. sinensis using large-scale sampling, and discusses the implications of ITS pseudogenes for fungal phylogenetic and evolutionary studies. Our results demonstrate a wide distribution of ITS pseudogenes amongst different geographic populations, and indicate how ITS pseudogenes can contribute to the reconstruction of the evolutionary history of the species.

Seasonal Variation and Crop Sequences Shape the Structure of Bacterial Communities in Cysts of Soybean Cyst Nematode.

Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the number 1 pathogen of the important economic crop soybean. Bacteria represent potential biocontrol agents of the SCN, but few studies have characterized the dynamics of bacterial communities associated with cysts under different crop rotation sequences. The bacterial communities in SCN cysts in a long-term soybean-corn crop rotation experiment were investigated over 2 years. The crop sequences included long-term soybean monoculture (Ss), years 1-5 of soybean following 5 years corn (S1-S5), years 1 and 2 of corn following 5 years soybean (C1 and C2), and soybean-corn annual rotation (Sa and Ca). The bacterial 16S rRNA V4 region was amplified from DNA isolated from SCN cysts collected in spring at planting, midseason (2 months later), and fall at harvest and sequenced on the Illumina MiSeq platform. The SCN cyst microbiome was dominated by Proteobacteria followed by Actinobacteria, Bacteroidetes, and Verrucomicrobia. The bacterial community composition was influenced by both crop sequence and season. Although differences by crop sequence were not significant in the spring of each year, bacterial communities in cysts from annual rotation (Sa and Ca) or crop sequences of early years of monoculture following a 5-year rotation of the alternate crop (S1 and C1) became rapidly differentiated by crop over a single growing season. In the fall, genera of cyst bacteria associated with soybean crop sequences included Rhizobacter, Leptothrix, Cytophaga, Chitinophaga, Niastella, Streptomyces, and Halangium. The discovery of diverse bacterial taxa in SCN cysts and their dynamics across crop rotation sequences provides invaluable information for future development of biological control of the SCN.

Module evolution and substrate specificity of fungal nonribosomal peptide synthetases involved in siderophore biosynthesis.

BACKGROUND: Most filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). While genes encoding the majority of NRPSs are intermittently distributed across the fungal kingdom, those encoding ferrichrome synthetase NRPSs, responsible for biosynthesis of ferrichrome siderophores, are conserved, which offers an opportunity to trace their evolution and the genesis of their multimodular domain architecture. Furthermore, since the chemistry of many ferrichromes is known, the biochemical and structural 'rules' guiding NRPS substrate choice can be addressed using protein structural modeling and evolutionary approaches. RESULTS: A search of forty-nine complete fungal genome sequences revealed that, with the exception of Schizosaccharomyces pombe, none of the yeast, chytrid, or zygomycete genomes contained a candidate ferrichrome synthetase. In contrast, all filamentous ascomycetes queried contained at least one, while presence and numbers in basidiomycetes varied. Genes encoding ferrichrome synthetases were monophyletic when analyzed with other NRPSs. Phylogenetic analyses provided support for an ancestral duplication event resulting in two main lineages. They also supported the proposed hypothesis that ferrichrome synthetases derive from an ancestral hexamodular gene, likely created by tandem duplication of complete NRPS modules. Recurrent losses of individual domains or complete modules from this ancestral gene best explain the diversity of extant domain architectures observed. Key residues and regions in the adenylation domain pocket involved in substrate choice and for binding the amino and carboxy termini of the substrate were identified. CONCLUSION: Iron-chelating ferrichrome synthetases appear restricted to fission yeast, filamentous ascomycetes, and basidiomycetes and fall into two main lineages. Phylogenetic analyses suggest that loss of domains or modules led to evolution of iterative biosynthetic mechanisms that allow flexibility in biosynthesis of the ferrichrome product. The 10 amino acid NRPS code, proposed earlier, failed when we tried to infer substrate preference. Instead, our analyses point to several regions of the binding pocket important in substrate choice and suggest that two positions of the code are involved in substrate anchoring, not substrate choice.

Mycobiome of Cysts of the Soybean Cyst Nematode Under Long Term Crop Rotation.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe (Phylum Nematoda), is a major pathogen of soybean. It causes substantial yield losses worldwide and is difficult to control because the cyst protects the eggs which can remain viable for nearly a decade. Crop rotation with non-host crops and use of biocontrol organisms such as fungi and bacteria offer promising approaches, but remain hampered by lack of knowledge of the biology of nematode parasitic organisms. We used a high-throughput metabarcoding approach to characterize fungal communities associated with the SCN cyst, a microenvironment in soil that may harbor both nematode parasites and plant pathogens. SCN cysts were collected from a long-term crop rotation experiment in Southeastern Minnesota at three time points over two growing seasons to characterize diversity of fungi inhabiting cysts and to examine how crop rotation and seasonal variation affects fungal communities. A majority of fungi in cysts belonged to Ascomycota and Basidiomycota, but the presence of several early diverging fungal subphyla thought to be primarily plant and litter associated, including Mortierellomycotina and Glomeromycotina (e.g., arbuscular mycorrhizal fungi), suggests a possible role as nematode egg parasites. Species richness varied by both crop rotation and season and was higher in early years of crop rotation and in fall at the end of the growing season. Crop rotation and season also impacted fungal community composition and identified several classes of fungi, including Eurotiomycetes, Sordariomycetes, and Orbiliomycetes (e.g., nematode trapping fungi), with higher relative abundance in early soybean rotations. The relative abundance of several genera was correlated with increasing years of soybean. Fungal communities also varied by season and were most divergent at midseason. The percentage of OTUs assigned to Mortierellomycotina_cls_Incertae_sedis and Sordariomycetes increased at midseason, while Orbiliomycetes decreased at midseason, and Glomeromycetes increased in fall. Ecological guilds of fungi containing an animal-pathogen lifestyle, as well as potential egg-parasitic taxa previously isolated from parasitized SCN eggs, increased at midseason. The animal pathogen guilds included known (e.g., Pochonia chlamydosporia) and new candidate biocontrol organisms. This research advances knowledge of the ecology of nematophagous fungi in agroecosystems and their use as biocontrol agents of the SCN.

Transcriptional responses of soybean roots to colonization with the root endophytic fungus Piriformospora indica reveals altered phenylpropanoid and secondary metabolism.

Piriformospora indica, a root endophytic fungus, has been shown to enhance biomass production and confer tolerance to various abiotic and biotic stresses in many plant hosts. A growth chamber experiment of soybean (Glycine max) colonized by P. indica compared to uninoculated control plants showed that the fungus significantly increased shoot dry weight, nutrient content, and rhizobial biomass. RNA-Seq analyses of root tissue showed upregulation of 61 genes and downregulation of 238 genes in colonized plants. Gene Ontology (GO) enrichment analyses demonstrated that upregulated genes were most significantly enriched in GO categories related to lignin biosynthesis and regulation of iron transport and metabolism but also mapped to categories of nutrient acquisition, hormone signaling, and response to drought stress. Metabolic pathway analysis revealed upregulation of genes within the phenylpropanoid and derivative pathways such as biosynthesis of monolignol subunits, flavonoids and flavonols (luteolin and quercetin), and iron scavenging siderophores. Highly enriched downregulated GO categories included heat shock proteins involved in response to heat, high-light intensity, hydrogen peroxide, and several related to plant defense. Overall, these results suggest that soybean maintains an association with this root endosymbiotic fungus that improves plant growth and nutrient acquisition, modulates abiotic stress, and promotes synergistic interactions with rhizobia.

Two genomes are better than one: history, genetics, and biotechnological applications of fungal heterokaryons.

Heterokaryosis is an integral part of the parasexual cycle used by predominantly asexual fungi to introduce and maintain genetic variation in populations. Research into fungal heterokaryons began in 1912 and continues to the present day. Heterokaryosis may play a role in the ability of fungi to respond to their environment, including the adaptation of arbuscular mycorrhizal fungi to different plant hosts. The parasexual cycle has enabled advances in fungal genetics, including gene mapping and tests of complementation, dominance, and vegetative compatibility in predominantly asexual fungi. Knowledge of vegetative compatibility groups has facilitated population genetic studies and enabled the design of innovative methods of biocontrol. The vegetative incompatibility response has the potential to be used as a model system to study biological aspects of some human diseases, including neurodegenerative diseases and cancer. By combining distinct traits through the formation of artificial heterokaryons, fungal strains with superior properties for antibiotic and enzyme production, fermentation, biocontrol, and bioremediation have been produced. Future biotechnological applications may include site-specific biocontrol or bioremediation and the production of novel pharmaceuticals.