Evaluation of genetic diversity, haplotype, and virulence of Fusarium oxysporum f. sp. vasinfectum field isolates from Alabama.

The United States is the third largest producer of cotton and the largest exporter of cotton globally. Fusarium wilt, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum (Fov), was estimated to cause a $21 million cotton yield loss in 2022. Historically, Alabama was an important producer of cotton in the southeastern United States and was the first state in which Fusarium wilt on cotton was described. To assess the genetic diversity of Fov field isolates in Alabama, 118 field isolates were collected from six counties across the state from 2014 to 2016. Phylogenetic analysis using TEF1 and RPB2 placed the Fov field isolates into 18 haplotypes. Upon profiling the Tfo1 transposon insertion in the NAT gene, it was determined that no race 4 isolates were recovered in Alabama. Representatives of all field isolate haplotypes caused disease on Upland cotton variety Rowden in a hydroponic test tube assay. Two haplotype A isolates were the most aggressive isolates recovered and haplotype A isolate TF1 was more aggressive than the race 4 isolate 89-1A on Upland cotton and had similar symptom severity on Pima cotton. Karyotype profiling indicted an abundance of small chromosomes characteristic of karyotypes that include accessory chromosomes, with considerable variability between isolates. Collectively, our study indicates that Fov isolates from Alabama are genetically diverse which may have been promoted by its persistence in cotton fields.

Key Challenges in Plant Pathology in the Next Decade.

Plant diseases significantly impact food security and food safety. It was estimated that food production needs to increase by 50% to feed the projected 9.3 billion people by 2050. Yet, plant pathogens and pests are documented to cause up to 40% yield losses in major crops, including maize, rice, and wheat, resulting in annual worldwide economic losses of approximately US$220 billion. Yield losses due to plant diseases and pests are estimated to be 21.5% (10.1 to 28.1%) in wheat, 30.3% (24.6 to 40.9%) in rice, and 22.6% (19.5 to 41.4%) in maize. In March 2023, The American Phytopathological Society (APS) conducted a survey to identify and rank key challenges in plant pathology in the next decade. Phytopathology subsequently invited papers that address those key challenges in plant pathology, and these were published as a special issue. The key challenges identified include climate change effect on the disease triangle and outbreaks, plant disease resistance mechanisms and its applications, and specific diseases including those caused by Candidatus Liberibacter spp. and Xylella fastidiosa. Additionally, disease detection, natural and man-made disasters, and plant disease control strategies were explored in issue articles. Finally, aspects of open access and how to publish articles to maximize the Findability, Accessibility, Interoperability, and Reuse of digital assets in plant pathology were described. Only by identifying the challenges and tracking progress in developing solutions for them will we be able to resolve the issues in plant pathology and ultimately ensure plant health, food security, and food safety.

Diversity of Fusarium community assembly shapes mycotoxin accumulation of diseased wheat heads.

Fusarium head blight (FHB) is a major disease worldwide on cultivated cereals, caused by several Fusarium species. FHB can cause not only yield reduction but also accumulation of mycotoxins in the grain contaminating the food supply. Much of the earlier research has focused on Fusarium pathogenesis, conditions required for disease development and toxin accumulation, and FHB management. However, the Fusarium community composition within the micro-habitat of a single diseased wheat head in the field has had limited investigation. Similarly, the relationship between the Fusarium community structure and mycotoxin accumulation within diseased heads remains unclear. In the present study, we investigated the Fusarium community in diseased heads sampled from different geographical sites in China. Several sites in Shandong province formed a transitional region which contained highly variable profiles of Fusarium OTUs, where a single diseased head could contain more than 10 Fusarium OTUs. Mycotoxin accumulation was independent of geographical properties, however, deoxynivalenol, 15-acetyldeoxynivalenol and zearalenone concentrations showed a significant negative correlation with Fusarium diversity on diseased heads while a significant positive correlation between nivalenol concentration and Fusarium diversity was observed. Taken together, the Fusarium OTU diversity within diseased heads in the field significantly influences mycotoxin accumulation, providing an important point to consider in FHB disease management and mycotoxin research.

Impact of osmotic stress on production, morphology, and fitness of Beauveria bassiana blastospores.

Beauveria bassiana is a cosmopolitan entomopathogenic fungus that can infect over 1000 insect species. During growth inside the host, B. bassiana transitions from hyphal to yeast-like unicellular growth as blastospores. Blastospores are well suited as an active ingredient in biopesticides due to their ease of production by liquid fermentation. Herein, we investigated the impact of hyperosmotic growth environments mediated by ionic and non-ionic osmolytes on two strains of B. bassiana (ESALQ1432 and GHA) relevant to growth morphology, blastospore production, desiccation tolerance, and insecticidal activity. Polyethylene glycol (PEG200) increased osmotic pressure in submerged cultures leading to decreased blastospore size but higher blastospore yields for one strain. Morphologically, decreased blastospore size was linked to increased osmotic pressure. However, smaller blastospores from PEG200 supplemented cultures after air-drying exhibited delayed germination. Ionic osmolytes (NaCl and KCl) generated the same osmotic pressure (2.5-2.7 MPa) as 20% glucose and boosted blastospore yields (> 2.0 × 109 blastospores mL-1). Fermentation performed in a bench-scale bioreactor consistently promoted high blastospore yields when using NaCl (2.5 MPa) amended media within 3 days. Mealworm larvae (Tenebrio molitor) were similarly susceptible to NaCl-grown blastospores and aerial conidia in a dose-time-dependent manner. Collectively, these results demonstrate the use of hyperosmotic liquid culture media in triggering enhanced yeast-like growth by B. bassiana. Understanding the role of osmotic pressure on blastospore formation and fitness will hasten the development of viable commercial fungal biopesticides. KEY POINTS: • Osmotic pressure plays a critical role in submerged fermentation of B. bassiana. • Ionic/non-ionic osmolytes greatly impact blastospore morphology, fitness, and yield. • Desiccation tolerance and bioefficacy of blastospores are affected by the osmolyte.

Rapid and Sensitive Detection of Verticillium dahliae from Complex Samples Using CRISPR/Cas12a Technology Combined with RPA.

Verticillium wilt is primarily caused by the fungus Verticillium dahliae and represents one of the most important worldwide soilborne plant diseases. The causal agent can be spread by microsclerotia and conidia attached to seeds during national/international trade or in soil between fields. Consequently, accurate, sensitive, and rapid detection of V. dahliae from complex samples is critical for restricting entry of the pathogen to a new region/environment and enforcing early management of Verticillium wilt. Based on CRISPR/Cas12a and recombinant polymerase amplification (RPA) technologies, we developed an accurate, sensitive, and rapid detection method for V. dahliae with paper-based lateral flow strips. A highly efficient and specific CRISPR RNA (crRNA) was designed for the GAPDH gene of V. dahliae and was validated using several closely related Verticillium spp. Excluding the time required for the DNA extraction from the complex samples, a minimum of 40 min was required for the detection time. The RPA-CRISPR/Cas12a detection system had a lower detection limit of ∼10 copies of genomic DNA per reaction and was able to successfully detect as little as one microsclerotium per gram of soil. In addition, field samples displaying symptoms suggestive of V. dahliae were able to be positively identified for the presence of V. dahliae. Taken together, this study broadens the applications of CRISPR/Cas12a nucleic acid detection to soilborne crop diseases and will contribute to the future development of field-deployable diagnostic tools.

Development of genetic markers for reproductive management of toucans.

Retention of genetic diversity in successive generations is key to successful ex situ programs and will become increasingly important to restore wild populations of threatened animals. When animal genealogy is partly unknown or gaps exist in studbook records, the application of molecular resources facilitates informed breeding. Here, we apply molecular resources to an ex situ breeding population of toucans (Ramphastidae), a bird family zoos commonly maintain. Toucans face population declines from illegal poaching and habitat degradation. We developed novel microsatellite markers using blood samples from 15 Keel-billed Toucans (Ramphastos sulfuratus Lesson 1830). Parentage of two individuals was known a priori, but possible sibship among 13 putative founders-including the parents-was unknown. We compared available avian heterologous and novel microsatellite markers to recover known relationships and reconstruct sibship. Eight of 61 heterologous markers amplified consistently and were polymorphic, but less so than the 18 novel markers. Known sibship (and three sibling pairs whose relatedness was unknown a priori) and paternity-though not maternity except in one case-were well-recovered using both likelihood and pairwise relatedness methods, when incorporating novel but not heterologous markers. Zoo researchers seeking microsatellite primer sets for their breeding toucan populations will likely benefit from our heterologous markers, which can be leveraged both to assess relatedness and select breeding pairs. We recommend that zoo biologists rely on species-specific primers and not optimize heterologous primers for toucan species without molecular resources. We conclude with a brief discussion of modern genotyping methods of interest to zoo researchers.

How Phylogenetics Can Elucidate the Chemical Ecology of Poison Frogs and Their Arthropod Prey.

The sequestration by neotropical poison frogs (Dendrobatidae) of an amazing array of defensive alkaloids from oribatid soil mites has motivated an exciting research theme in chemical ecology, but the details of mite-to-frog transfer remain hidden. To address this, McGugan et al. (2016, Journal of Chemical Ecology 42:537-551) used the little devil poison frog (Oophaga sylvatica) and attempted to simultaneously characterize the prey mite alkaloids, the predator skin alkaloids, and identify the mites using DNA sequences. Heethoff et al. (2016, Journal of Chemical Ecology 42:841-844) argued that none of the mite families to which McGugan et al. allocated the prey was thought to possess alkaloids. Heethoff et al. concluded from analyses including additional sequences that the mite species were unlikely to be close relatives of the defended mites. We re-examine this by applying more appropriate phylogenetic methods to broader and denser taxonomic samples of mite sequences using the same gene (CO1). We found, over trees based on CO1 datasets, only weak support (except in one case) for branches critical to connecting the evolution of alkaloid sequestration with the phylogeny of mites. In contrast, a well-supported analysis of the 18S ribosomal gene suggests at least two independent evolutionary origins of oribatid alkaloids. We point out impediments in the promising research agenda, namely a paucity of genetic, chemical, and taxonomic information, and suggest how phylogenetics can elucidate at a broader level the evolution of chemical defense in prey arthropods, sequestration by predators, and the impact of alkaloids on higher-order trophic interactions.

Genome Resource: Draft Genome of Fusarium avenaceum, Strain F156N33, Isolated from the Atmosphere Above Virginia and Annotated Based on RNA Sequencing Data.

Fusarium avenaceum is a filamentous fungus commonly associated with plants and soil. It is a causal agent of Fusarium head blight (FHB) on maize and small-grain cereals and blights on other plant species, and is one of the very few fungal species known to have ice nucleation activity (i.e., it catalyzes ice formation). Here, we report the draft genome of the ice-nucleation-active F. avenaceum strain F156N33 isolated from the atmosphere above Virginia. The genome assembly is 41,175,306 bp long, consists of 214 contigs, and is predicted to encode 11,233 proteins, which were annotated using RNA-sequencing data obtained from the same strain.

An In Vitro Co-Culture System for Rapid Differential Response to Fusarium oxysporum f. sp. vasinfectum Race 4 in Three Cotton Cultivars.

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a devastating fungus pathogen that causes Fusarium wilt in both domesticated cotton species, Gossypium hirsutum (Upland) and G. barbadense (Pima). Greenhouse and field-based pathogenicity assays can be a challenge because of nonuniform inoculum levels, the presence of endophytes, and varying environmental factors. Therefore, an in vitro coculture system was designed to support the growth of both domesticated cotton species and FOV4 via an inert polyphenolic foam substrate with a liquid medium. A Fusarium wilt-susceptible Pima cotton cultivar, G. barbadense 'GB1031'; a highly resistant Pima cotton cultivar, G. barbadense 'DP348RF'; and a susceptible Upland cotton cultivar, G. hirsutum 'TM-1', were evaluated for 30 days during coculture with FOV4 in this foam-based system. Thirty days after inoculation, disease symptoms were more severe in both susceptible cultivars, which displayed higher percentages of foliar damage, and greater plant mortality than observed in 'DP348RF', the resistant Pima cotton cultivar. This foam-based in vitro system may be useful for screening cotton germplasm for resistance to a variety of fungus pathogens and may facilitate the study of biotic interactions in domesticated cotton species under controlled environmental conditions.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

The Genome Sequence of Five Genotypes of Fusarium oxysporum f. sp. vasinfectum: A Resource for Studies on Fusarium Wilt of Cotton.

Fusarium oxysporum f. sp. vasinfectum is an important plant pathogen responsible for vascular wilt disease on cotton. Members of this group are known to carry supernumerary chromosomes that encode virulence factors. We sequenced the genomes of five F. oxysporum f. sp. vasinfectum isolates, including the genome of a representative of the highly virulent genotype race 4, at a high coverage to assemble reference-quality genomes. These genomes provide a necessary resource for comparative genomic analyses to identify genes or genome features that are involved in pathogenicity on cotton and may ultimately be used to identify improved management strategies.

CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum.

Fusarium oxysporum is an economically important pathogen that widely exists in the environment and is capable of causing serious problems in crop production and animal/human health. One important step for characterization of a fungal protein with an unknown function is to determine its subcellular localization within the cell. To facilitate the study of important functional regulators or key virulence factors, we developed a CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). The HITI strategy was able to facilitate integration of a large DNA fragment, ∼8 kb in length, into the genome of F. oxysporum at the sgRNA cleavage site, and was used to insert a C-terminal 3×sGFP tag to the FoCHS5 gene and a N-terminal mCherry tag to the FoSSO2 gene. The HDRI strategy was used to tag the paralogous gene, FoSSO1, with a C-terminal mCherry marker. FoChs5-3×sGFP localized to conidia, some septa, and fungal tips. A majority of the FoSso1-mCherry was distributed in the conidia, septum, and hyphae that were distal from the fungal tips. While FoSso1-mCherry showed a very weak fluorescent signal at the fungal tips, mCherry-FoSso2 accumulated in the plasma membrane of conidia, germlings, fungal tips, hyphae, and phialides, suggesting FoSSO1 and FoSSO2 are regulated differently during fungal development. These results indicate this EGT system is efficient and can be another molecular tool to resolve the function(s) of proteins and infection strategies of F. oxysporum.

Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein complexes.

The Fusarium oxysporum species complex (FOSC) is an economically important group of pathogenic filamentous fungi that are able to infect both animals and plants. Reverse genetic techniques, including gene disruption/deletion methods, to study these fungi are available although limitations exist resulting in decreased efficiency. Herein we describe a gene editing system developed using a F. oxysporum-optimized Cas9 ribonucleoprotein (RNP) and protoplast transformation method. The Cas9 protein and sgRNA were assembled to form a stable RNP in vitro and this complex was transferred into fungal protoplasts for gene editing with PEG-mediated transformation. In order to determine if the Cas9 RNP system is functional in the FOSC protoplasts and assess the efficacy of the system, two genes, URA5 and URA3, were selected for targeted disruption generating uracil auxotroph mutants that are resistant to 5-fluoroorotic acid, 5-FOA. In addition, a gene in a secondary metabolite biosynthetic cluster, the ortholog of BIK1, was mutated using this system and the maximum efficiency of this gene disruption was about 50%. Further analysis of the bik1 mutant confirmed that this polyketide synthase was involved in the synthesis of the red pigment, bikaverin. The mutants generated in this study displayed the strong expected phenotypes, demonstrating this F. oxysporum-optimized CRISPR/Cas9 system is stable and can efficiently disrupt the genes of interest.

Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds.

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z' factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 µg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Passive accumulation of alkaloids in non-toxic frogs challenges paradigms of the origins of acquired chemical defenses.

Understanding the origins of novel, complex phenotypes is a major goal in evolutionary biology. Poison frogs of the family Dendrobatidae have evolved the novel ability to acquire alkaloids from their diet for chemical defense at least three times. However, taxon sampling for alkaloids has been biased towards colorful species, without similar attention paid to inconspicuous ones that are often assumed to be undefended. As a result, our understanding of how chemical defense evolved in this group is incomplete. Here we provide new data showing that, in contrast to previous studies, species from each undefended poison frog clade have measurable yet low amounts of alkaloids. We confirm that undefended dendrobatids regularly consume mites and ants, which are known sources of alkaloids. Further, we confirm the presence of alkaloids in two putatively non-toxic frogs from other families. Our data suggest the existence of a phenotypic intermediate between toxin consumption and sequestration-passive accumulation-that differs from active sequestration in that it involves no derived forms of transport and storage mechanisms yet results in low levels of toxin accumulation. We discuss the concept of passive accumulation and its potential role in the origin of chemical defenses in poison frogs and other toxin-sequestering organisms.

One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

Inventory of the Secondary Metabolite Biosynthetic Potential of Members within the Terminal Clade of the Fusarium solani Species Complex.

The Fusarium solani species complex (FSSC) constitutes at least 77 phylogenetically distinct species including several agriculturally important and clinically relevant opportunistic pathogens. As with other Fusaria, they have been well documented to produce many secondary metabolites-compounds that are not required for the fungus to grow or develop but may be beneficial to the organism. An analysis of ten genomes from fungi within the terminal clade (clade 3) of the FSSC revealed each genome encoded 35 (F. cucurbitcola) to 48 (F. tenucristatum) secondary metabolite biosynthetic gene clusters (BGCs). A total of seventy-four different BGCs were identified from the ten FSSC genomes including seven polyketide synthases (PKS), thirteen nonribosomal peptide synthetases (NRPS), two terpene synthase BGCs, and a single dimethylallytryptophan synthase (DMATS) BGC conserved in all the genomes. Some of the clusters that were shared included those responsible for producing naphthoquinones such as fusarubins, a red pigmented compound, squalestatin, and the siderophores malonichrome, ferricrocin, and triacetylfusarinine. Eight novel NRPS and five novel PKS BGCs were identified, while BGCs predicted to produce radicicol, gibberellin, and fusaoctaxin were identified, which have not previously described in members of the FSSC. The diversity of the secondary metabolite repertoire of the FSSC may contribute to the expansive host range of these fungi and their ability to colonize broad habitats.

Polymerase chain reaction-based assays for the diagnosis of invasive fungal infections.

Currently accepted fungal diagnostic techniques, such as culture, biopsy, and serology, lack rapidity and efficiency. Newer diagnostic methods, such as polymerase chain reaction (PCR)-based assays, have the potential to improve fungal diagnostics in a faster, more sensitive, and specific manner. Preliminary data indicate that, when PCR-based fungal diagnostic assays guide antifungal therapy, they may lower patient mortality and decrease unnecessary antifungal treatment, improving treatment-associated costs and avoiding toxicity. Moreover, newer PCR techniques can identify antifungal resistance DNA loci, but the clinical correlation between those loci and clinical failure has to be studied further. In addition, future studies need to focus on the implementation of PCR techniques in clinical decision making and on combining them with other diagnostic tests. A consensus on the standardization of PCR techniques, along with validation from large prospective studies, is necessary to allow widespread adoption of these assays.