Members of the Fusarium oxysporum Complex Causing Wilt Symptoms in Medical Cannabis in Israel, Italy, and North America Comprise a Polyphyletic Assemblage.

Members of the Fusarium oxysporum complex are ubiquitous soilborne fungal pathogens causing wilt diseases in various plant hosts. Fusarium oxysporum (Fo) f. sp. cannabis was first reported causing wilt disease in hemp in Italy in 1962. To date, Fusarium wilt continues to cause concern in industrial and medicinal cannabis cultivation worldwide. During a 3-year period (2018 to 2021), Fo strains were isolated from medical cannabis plants (Cannabis sativa) exhibiting wilt symptoms that were cultivated in numerous commercial farms in Israel. A diverse set of these strains was subjected to molecular phylogenetic analyses to assess their genetic diversity and to compare them with other f. sp. cannabis isolates included in prior studies. Maximum likelihood bootstrap analysis of a partial translation elongation factor (TEF1) dataset, which included 24 f. sp. cannabis sequences, revealed that the 11 strains from Israel comprised five TEF1 haplotypes. Two of the haplotypes from Israel were identical to isolates previously reported from British Columbia and California and British Columbia and Ontario. Overall, the 24 f. sp. cannabis sequences included 12 unique TEF1 haplotypes. These were phylogenetically diverse, suggesting that pathogenicity to C. sativa may have evolved independently within the F. oxysporum complex. Pathogenicity tests of the Israeli strains were confirmed by Koch's postulates assays. Strains of the five different f. sp. cannabis TEF1 haplotypes all caused wilt in cannabis seedlings but with varying levels of aggressiveness. The same isolates that originated from asymptomatic infected mother plants were found in wilted cuttings indicating that the pathogen can be spread via propagation material.

Myxomatous mitral valve disease in Miniature Schnauzers and Yorkshire Terriers: 134 cases (2007-2016).

OBJECTIVE: To characterize features of myxomatous mitral valve disease (MMVD) in Miniature Schnauzers and Yorkshire Terriers. ANIMALS: 69 Miniature Schnauzers and 65 Yorkshire Terriers, each with MMVD. PROCEDURES: Medical record data for each dog were collected; the study period was January 2007 through December 2016. If available, radiographic data were evaluated, and a vertebral heart scale score was assigned for each dog. Statistical analysis was performed with Student t and Fisher exact tests. RESULTS: Compared with Yorkshire Terriers, the prevalence of MMVD was significantly higher in Miniature Schnauzers and affected dogs were significantly younger at the time of diagnosis. Miniature Schnauzers were significantly more likely to have mitral valve prolapse and syncope, compared with Yorkshire Terriers. Yorkshire Terriers were significantly more likely to have coughing and have had previous or current treatment with cardiac medications, compared with Miniature Schnauzers. There was no statistical difference between breeds with regard to abnormally high vertebral heart scale scores or radiographic evidence of congestive heart failure. CONCLUSIONS AND CLINICAL RELEVANCE: With regard to MMVD, features of the disease among Miniature Schnauzers and Yorkshire Terriers were similar, but there were also a few discernable differences between these 2 breeds and from historical findings for dogs with MMVD of other breeds. Clinical signs at the time of diagnosis differed between the 2 breeds, which may have reflected concurrent breed-specific conditions (sick sinus syndrome or airway disease [eg, tracheal collapse]). Future work should include prospective studies to provide additional information regarding the natural progression of MMVD in these dog breeds.

Resolving the Mortierellaceae phylogeny through synthesis of multi-gene phylogenetics and phylogenomics.

Early efforts to classify Mortierellaceae were based on macro- and micromorphology, but sequencing and phylogenetic studies with ribosomal DNA (rDNA) markers have demonstrated conflicting taxonomic groupings and polyphyletic genera. Although some taxonomic confusion in the family has been clarified, rDNA data alone is unable to resolve higher level phylogenetic relationships within Mortierellaceae. In this study, we applied two parallel approaches to resolve the Mortierellaceae phylogeny: low coverage genome (LCG) sequencing and high-throughput, multiplexed targeted amplicon sequencing to generate sequence data for multi-gene phylogenetics. We then combined our datasets to provide a well-supported genome-based phylogeny having broad sampling depth from the amplicon dataset. Resolving the Mortierellaceae phylogeny into monophyletic groups led to the definition of 14 genera, 7 of which are newly proposed. Low-coverage genome sequencing proved to be a relatively cost-effective means of generating a well-resolved phylogeny. The multi-gene phylogenetics approach enabled much greater sampling depth and breadth than the LCG approach, but was unable to resolve higher-level organization of groups. We present this work to resolve some of the taxonomic confusion and provide a genus-level framework to empower future studies on Mortierellaceae diversity, biology, and evolution.

Pseudoflowers produced by Fusarium xyrophilum on yellow-eyed grass (Xyris spp.) in Guyana: A novel floral mimicry system?

Pseudoflower formation is arguably the rarest outcome of a plant-fungus interaction. Here we report on a novel putative floral mimicry system in which the pseudoflowers are composed entirely of fungal tissues in contrast to modified leaves documented in previous mimicry systems. Pseudoflowers on two perennial Xyris species (yellow-eyed grass, X. setigera and X. surinamensis) collected from savannas in Guyana were produced by Fusarium xyrophilum, a novel Fusarium species. These pseudoflowers mimic Xyris flowers in gross morphology and are ultraviolet reflective. Axenic cultures of F. xyrophilum produced two pigments that had fluorescence emission maxima in light ranges that trichromatic insects are sensitive to and volatiles known to attract insect pollinators. One of the volatiles emitted by F. xyrophilum cultures (i.e., 2-ethylhexanol) was also detected in the head space of X. laxifolia var. iridifolia flowers, a perennial species native to the New World. Results of microscopic and PCR analyses, combined with examination of gross morphology of the pseudoflowers, provide evidence that the fungus had established a systemic infection in both Xyris species, sterilized them and formed fungal pseudoflowers containing both mating type idiomorphs. Fusarium xyrophilum cultures also produced the auxin indole-3-acetic acid (IAA) and the cytokinin isopentenyl adenosine (iPR). Field observations revealed that pseudoflowers and Xyris flowers were both visited by bees. Together, the results suggest that F. xyrophilum pseudoflowers are a novel floral mimicry system that attracts insect pollinators, via visual and olfactory cues, into vectoring its conidia, which might facilitate outcrossing of this putatively heterothallic fungus and infection of previously uninfected plants.

Maternal mitochondrial inheritance in two Fusarium pathogens of prickly ash (Zanthoxylum bungeanum) in northern China.

Mitochondrial inheritance in Fusarium zanthoxyli and F. continuum, two canker-inducing pathogens of prickly ash (Zanthoxylum bungeanum) in northern China, was investigated by genotyping ascospore progeny obtained from laboratory crosses. Polymorphic regions of the mitochondrial genomes (mitogenomes) that contained indels and single-nucleotide polymorphisms (SNPs) were identified via comparative analyses of the complete mitogenomes of the parents used in the intraspecific crosses. A reciprocal genetic cross of F. zanthoxyli NRRL 66714 × NRRL 66285, and a separate cross of F. continuum ♀ NRRL 66286 × â™‚ NRRL 66218, revealed that mitochondria were only inherited from the maternal parent. In addition, the reciprocal cross demonstrated that mitochondrial inheritance is not linked to mating type. Gene order in the circular mitogenomes of the prickly ash pathogens was identical to that previously reported for other fusaria and members of the Hypocreales, except that the TRNL tRNAs were duplicated in F. zanthoxyli NRRL 66714. The genomes contained 14 polypeptide-encoding genes involved in oxidative respiration, one intron-encoded ribosomal protein (rps3) gene, two ribosomal RNA (rRNA) genes, and 26-28 tRNA genes. The F. zanthoxyli mitogenomes were 80.9 and 98.7 kb in length, whereas those of F. continuum were considerably shorter and nearly identical in length at 63.4 kb. The significant differences in mitogenome length were primarily due to variable numbers of introns and open reading frames (ORFs) encoding hypothetical proteins.

Molecular systematics of two sister clades, the Fusarium concolor and F. babinda species complexes, and the discovery of a novel microcycle macroconidium-producing species from South Africa.

Multilocus DNA sequence data were used to investigate species identity and diversity in two sister clades, the Fusarium concolor (FCOSC) and F. babinda species complexes. Of the 109 isolates analyzed, only 4 were received correctly identified to species and these included 1/46 F. concolor, 1/31 F. babinda, and 2/3 F. anguioides. The majority of the F. concolor and F. babinda isolates were received as F. polyphialidicum, which is a heterotypic synonym of the former species. Previously documented from South America, Africa, Europe, and Australia, our data show that F. concolor is also present in North America. The present study expands the known distribution of F. babinda in Australia to Asia, Europe, and North America. The molecular phylogenetic results support the recognition of a novel Fusarium species within the FCOSC, which is described and illustrated here as F. austroafricanum, sp. nov. It was isolated as an endophyte of kikuyu grass associated with a putative mycotoxicosis of cattle and from plant debris in soil in South Africa. Fusarium austroafricanum is most similar morphologically to F. concolor and F. babinda but differs from the latter two species in producing (i) much longer macroconidia in which the apical cell is blunt to slightly papillate and the basal cell is only slightly notched and (ii) macroconidia via microcycle conidiation on water agar. BLASTn searches of the whole genome sequence of F. austroafricanum NRRL 53441 were conducted to predict mycotoxin potential, using genes known to be essential for the synthesis of several mycotoxins and biologically active metabolites. Based on the presence of intact gene clusters that confer the ability to synthesize mycotoxins and pigments, we analyzed cracked corn kernel cultures of F. austroafricanum via liquid chromatography-mass spectrometry (LC-MS) but failed to detect these metabolites in vitro.

Intraspecific adaptive radiation: Competition, ecological opportunity, and phenotypic diversification within species.

Intraspecific variation in resource-use traits can have profound ecological and evolutionary implications. Among the most striking examples are resource polymorphisms, where alternative morphs that utilize different resources evolve within a population. An underappreciated aspect of their evolution is that the same conditions that favor resource polymorphism-competition and ecological opportunity-might foster additional rounds of diversification within already existing morphs. We examined these issues in spadefoot toad tadpoles that develop into either a generalist "omnivore" or a specialist "carnivore" morph. Specifically, we assessed the morphological diversity of tadpoles from natural ponds and experimentally induced carnivores reared on alternative diets. We also surveyed natural ponds to determine if the strength of intramorph competition and the diversity and abundance of dietary resources (measures of ecological opportunity) influenced the diversity of within-morph variation. We found that five omnivore and four carnivore types were present in natural ponds; alternative diets led to shape differences, some of which mirrored variation in the wild; and both competition and ecological opportunity were associated with enhanced morphological diversity in natural ponds. Such fine-scale intraspecific variation might represent an underappreciated form of biodiversity and might constitute a crucible of evolutionary innovation and diversification.

Fusarium algeriense, sp. nov., a novel toxigenic crown rot pathogen of durum wheat from Algeria is nested in the Fusarium burgessii species complex.

A novel crown rot pathogen of wheat discovered during pathogen surveys in Algeria in 2014 and 2015 is formally described as Fusarium algeriense. Multilocus molecular phylogenetic data resolved the eight isolates of this pathogen as a genealogically exclusive species lineage in the F. burgessii species complex. The previously described species of this complex, F. burgessii and F. beomiforme, produce abundant chlamydospores in culture, and their optimal temperature for growth is 30 C. In comparison, F. algeriense did not produce chlamydospores under the conditions tested and its optimal temperature for growth is 25 C. Furthermore, F. algeriense differs from F. burgessii because it does not produce polyphialides and F. beomiforme, because it does not produce globose-to-napiform conidia in the aerial mycelium. Isolates of F. algeriense induced moderate crown rot on the susceptible spring wheat cultivar Norm in a temperature-controlled incubator. Fusarium burgessii and F. beomiforme, in contrast, only induced mild symptoms of this disease. BLASTn searches of the whole-genome sequence of F. algeriense strains NRRL 66647 and 66648, using homologs of genes that are responsible for synthesis of toxic secondary metabolites, indicated that they have the potential to produce several polyketide and non-ribosomal peptide-derived mycotoxins. However, moniliformin and 2-AOD-ol (2-amino-14,16-dimethyloctadecan-3-ol) were the only mycotoxins detected by liquid chromatography-mass spectrometry (LC-MS) analyses of strains cultivated in vitro on a solid medium. A polymerase chain reaction (PCR) assay for MAT idiomorph revealed that MAT1-1 and MAT1-2 strains of F. algeriense were present in Algeria, which suggests this pathogen might possess a heterothallic sexual reproductive mode.

Races of the Celery Pathogen Fusarium oxysporum f. sp. apii Are Polyphyletic.

Fusarium oxysporum species complex (FOSC) isolates were obtained from celery with symptoms of Fusarium yellows between 1993 and 2013 primarily in California. Virulence tests and a two-gene dataset from 174 isolates indicated that virulent isolates collected before 2013 were a highly clonal population of F. oxysporum f. sp. apii race 2. In 2013, new highly virulent clonal isolates, designated race 4, were discovered in production fields in Camarillo, California. Long-read Illumina data were used to analyze 16 isolates: six race 2, one of each from races 1, 3, and 4, and seven genetically diverse FOSC that were isolated from symptomatic celery but are nonpathogenic on this host. Analyses of a 10-gene dataset comprising 38 kb indicated that F. oxysporum f. sp. apii is polyphyletic; race 2 is nested within clade 3, whereas the evolutionary origins of races 1, 3, and 4 are within clade 2. Based on 6,898 single nucleotide polymorphisms from the core FOSC genome, race 3 and the new highly virulent race 4 are highly similar with Nei's Da = 0.0019, suggesting that F. oxysporum f. sp. apii race 4 evolved from race 3. Next generation sequences were used to develop PCR primers that allow rapid diagnosis of races 2 and 4 in planta.

Fusarium agapanthi sp. nov., a novel bikaverin and fusarubin-producing leaf and stem spot pathogen of Agapanthus praecox (African lily) from Australia and Italy.

This study was conducted to characterize a novel Fusarium species that caused leaf and stem spot on Agapanthus praecox (Agapanthus, African lily) in northern Italy and leaf rot and spot on the same host in Melbourne, Australia. Formally described as Fusarium agapanthi, this pathogen was analyzed using phenotypic, phytopathogenic, secondary metabolite, molecular phylogenetic and genomic data. Five strains were characterized, including one isolated in 1999 from symptomatic A. praecox in Saluzzo, Italy, and four in 2010 from diseased leaf tissue from the same host exhibiting leaf rot and spot symptoms in the Melbourne Gardens, Royal Botanic Gardens Victoria, Australia. Maximum parsimony and maximum likelihood molecular phylogenetic analyses of portions of six individual genes and the combined dataset all strongly supported F. agapanthi either as the earliest diverging genealogically exclusive lineage in the American Clade of the F. fujikuroi species complex, or alternatively a novel monotypic lineage sister to the American Clade. Koch's postulates were completed on dwarf blue- and large white-flowering varieties of A. praecox, where two isolates of F. agapanthi produced slowly spreading necrotic lesions when inoculated onto leaves and flower stems. Fusarium agapanthi is distinguished from other fusaria by the production of densely branched aerial conidiophores with polyphialides throughout the aerial mycelium on synthetic nutrient-poor agar. BLASTn searches of the F. agapanthi NRRL 31653 and NRRL 54464 (= VPRI 41787) genome sequences were conducted to predict sexual reproductive mode and mycotoxin potential. Results indicated that they possessed MAT1-2 and MAT1-1 idiomorphs, respectively, indicating that this species might be heterothallic. Furthermore, based on the presence of homologs of the bikaverin and fusarubin biosynthetic gene clusters in the F. agapanthi genomes, liquid chromatography-mass spectrometry analysis was conducted and confirmed production of these secondary metabolites in rice and corn kernel cultures of the fungus.

Fusarium pathogenomics.

Fusarium is a genus of filamentous fungi that contains many agronomically important plant pathogens, mycotoxin producers, and opportunistic human pathogens. Comparative analyses have revealed that the Fusarium genome is compartmentalized into regions responsible for primary metabolism and reproduction (core genome), and pathogen virulence, host specialization, and possibly other functions (adaptive genome). Genes involved in virulence and host specialization are located on pathogenicity chromosomes within strains pathogenic to tomato (Fusarium oxysporum f. sp. lycopersici) and pea (Fusarium 'solani' f. sp. pisi). The experimental transfer of pathogenicity chromosomes from F. oxysporum f. sp. lycopersici into a nonpathogen transformed the latter into a tomato pathogen. Thus, horizontal transfer may explain the polyphyletic origins of host specificity within the genus. Additional genome-scale comparative and functional studies are needed to elucidate the evolution and diversity of pathogenicity mechanisms, which may help inform novel disease management strategies against fusarial pathogens.

Chronic rhinofacial mucormycosis caused by Mucor irregularis (Rhizomucor variabilis) in India.

In this article, we describe a chronic case of rhinofacial mucormycosis caused by Mucor irregularis, formerly known as Rhizomucor variabilis var. variabilis, a rare mycotic agent in humans. The infection caused progressive destruction of the nasal septum and soft and hard palate, leading to collapse of the nose bridge and an ulcerative gaping hole. The mucoralean mold cultured from a nasal biopsy specimen was determined by multilocus DNA sequence data to be conspecific with M. irregularis.

The Ascomycota tree of life: a phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits.

We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.

Phylogeny of the Zygomycota based on nuclear ribosomal sequence data.

The Zygomycota is an ecologically heterogenous assemblage of nonzoosporic fungi comprising two classes, Zygomycetes and Trichomycetes. Phylogenetic analyses have suggested that the phylum is polyphyletic; two of four orders of Trichomycetes are related to the Mesomycetozoa (protists) that diverged near the fungal/animal split. Current circumscription of the Zygomycota includes only orders with representatives that produce zygospores. We present a molecular-based phylogeny including recognized representatives of the Zygomycetes and Trichomycetes with a combined dataset for nuclear rRNA 18S (SSU), 5.8S and 28S (LSU) genes. Tree reconstruction by Bayesian analyses suggests the Zygomycota is paraphyletic. Although 12 clades were identified only some of these correspond to the nine orders of Zygomycota currently recognized. A large superordinal clade, comprising the Dimargaritales, Harpellales, Kickxellales and Zoopagales, grouping together many symbiotic fungi, also is identified in part by a unique septal structure. Although Harpellales and Kickxellales are not monophyletic, these lineages are distinct from the Mucorales, Endogonales and Mortierellales, which appear more closely related to the Ascomycota + Basidiomycota + Glomeromycota. The final major group, the insect-associated Entomophthorales, appears to be polyphyletic. In the present analyses Basidiobolus and Neozygites group within Zygomycota but not with the Entomophthorales. Clades are discussed with special reference to traditional classifications, mapping morphological characters and ecology, where possible, as a snapshot of our current phylogenetic perspective of the Zygomycota.

Fusarium commune is a new species identified by morphological and molecular phylogenetic data.

Fusarium commune sp. nov. was isolated from soil and Pisum sativum in Denmark and several widespread locations within the northern hemisphere from diverse substrates including white pine, Douglas fir, carnation, corn, carrot, barley and soil. Fusarium commune is characterized by and distinguished from its putative sister taxon, the F. oxysporum complex, in having long, slender monophialides and polyphialides when cultured in the dark. Based on the combined DNA sequence data from translation elongation factor 1α (EF-1α) and the mitochondrial small subunit ribosomal DNA (mtSSU rDNA), the 15 isolates of F. commune analyzed formed a strongly supported clade closely related to but independent of the F. oxysporum and Gibberella fujikuroi species complexes.