Cold atmospheric plasma inactivates Aspergillus flavus and Fusarium keratoplasticum biofilms and conidia in vitro.

Introduction. Aspergillus flavus and Fusarium keratoplasticum are common causative pathogens of fungal keratitis (FK), a severe corneal disease associated with significant morbidity and vision loss. Escalating incidence of antifungal resistance to available antifungal drugs poses a major challenge to FK treatment. Cold atmospheric plasma (CAP) is a pioneering nonpharmacologic antimicrobial intervention that has demonstrated potential as a broad-spectrum antifungal treatment.Gap statement. Previous research highlights biofilm-associated resistance as a critical barrier to effective FK treatment. Although CAP has shown promise against various fungal infections, its efficacy against biofilm and conidial forms of FK pathogens remains inadequately explored.Aim. This study aims to investigate the antifungal efficacy of CAP against clinical fungal keratitis isolates of A. flavus and F. keratoplasticum in vitro.Methodology. Power parameters (22-27 kVpp, 300-400 Hz and 20-80 mA) of a dielectric barrier discharge CAP device were optimized for inactivation of A. flavus biofilms. Optimal applied voltage and total current were applied to F. keratoplasticum biofilms and conidial suspensions of A. flavus and F. keratoplasticum. The antifungal effect of CAP treatment was investigated by evaluating fungal viability through means of metabolic activity, c.f.u. enumeration (c.f.u. ml-1) and biofilm formation.Results. For both fungal species, CAP exhibited strong time-dependent inactivation, achieving greater than 80 % reduction in metabolic activity and c.f.u. ml-1 within 300 s or less, and complete inhibition after 600 s of treatment.Conclusion. Our findings indicate that CAP is a promising broad-spectrum antifungal intervention. CAP treatment effectively reduces fungal viability in both biofilm and conidial suspension cultures of A. flavus and F. keratoplasticum, suggesting its potential as an alternative treatment strategy for fungal keratitis.

Inactivation of siderophore iron-chelating moieties by the fungal wheat root symbiont Pyrenophora biseptata.

We investigated the ability of four plant and soil-associated fungi to modify or degrade siderophore structures leading to reduced siderophore iron-affinity in iron-limited and iron-replete cultures. Pyrenophora biseptata, a melanized fungus from wheat roots, was effective in inactivating siderophore iron-chelating moieties. In the supernatant solution, the tris-hydroxamate siderophore desferrioxamine B (DFOB) underwent a stepwise reduction of the three hydroxamate groups in DFOB to amides leading to a progressive loss in iron affinity. A mechanism is suggested based on the formation of transient ferrous iron followed by reduction of the siderophore hydroxamate groups during fungal high-affinity reductive iron uptake. P. biseptata also produced its own tris-hydroxamate siderophores (neocoprogen I and II, coprogen and dimerum acid) in iron-limited media and we observed loss of hydroxamate chelating groups during incubation in a manner analogous to DFOB. A redox-based reaction was also involved with the tris-catecholate siderophore protochelin in which oxidation of the catechol groups to quinones was observed. The new siderophore inactivating activity of the wheat symbiont P. biseptata is potentially widespread among fungi with implications for the availability of iron to plants and the surrounding microbiome in siderophore-rich environments.

PacBio high-throughput multi-locus sequencing reveals high genetic diversity in mushroom-forming fungi.

Multi-locus sequence data are widely used in fungal systematic and taxonomic studies to delimit species and infer evolutionary relationships. We developed and assessed the efficacy of a multi-locus pooled sequencing method using PacBio long-read high-throughput sequencing. Samples included fresh and dried voucher specimens, cultures and archival DNA extracts of Agaricomycetes with an emphasis on the order Cantharellales. Of the 283 specimens sequenced, 93.6% successfully amplified at one or more loci with a mean of 3.3 loci amplified. Our method recovered multiple sequence variants representing alleles of rDNA loci and single copy protein-coding genes rpb1, rpb2 and tef1. Within-sample genetic variation differed by locus and taxonomic group, with the greatest genetic divergence observed among sequence variants of rpb2 and tef1 from corticioid Cantharellales. Our method is a cost-effective approach for generating accurate multi-locus sequence data coupled with recovery of alleles from polymorphic samples and multi-organism specimens. These results have important implications for understanding intra-individual genomic variation among genetic loci commonly used in species delimitation of fungi.

Phase-Dependent Differential In Vitro and Ex Vivo Susceptibility of Aspergillus flavus and Fusarium keratoplasticum to Azole Antifungals.

Fungal keratitis (FK) is an invasive infection of the cornea primarily associated with Aspergillus and Fusarium species. FK is treated empirically with a limited selection of topical antifungals with varying levels of success. Though clinical infections are typically characterized by a dense network of mature mycelium, traditional models used to test antifungal susceptibility of FK isolates exclusively evaluate susceptibility in fungal cultures derived from asexual spores known as conidia. The purpose of this study was to characterize differences in fungal response when topical antifungal treatment is initiated at progressive phases of fungal development. We compared the efficacy of voriconazole and luliconazole against in vitro cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. A porcine cadaver corneal model was used to compare antifungal efficacy of voriconazole and luliconazole in ex vivo tissue cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. Our results demonstrate phase-dependent susceptibility of both A. flavus and F. keratoplasticum to both azoles in vitro as well as ex vivo. We conclude that traditional antifungal susceptibility testing with conidial suspensions does not correlate with fungal susceptibility in cultures of a more advanced developmental phase. A revised method of antifungal susceptibility testing that evaluates hyphal susceptibility may better predict fungal response in the clinical setting where treatment is often delayed until days after the initial insult.

Comparative genomic analysis reveals contraction of gene families with putative roles in pathogenesis in the fungal boxwood pathogens Calonectria henricotiae and C. pseudonaviculata.

BACKGROUND: Boxwood blight disease caused by Calonectria henricotiae and C. pseudonaviculata is of ecological and economic significance in cultivated and native ecosystems worldwide. Prior research has focused on understanding the population genetic and genomic diversity of C. henricotiae and C. pseudonaviculata, but gene family evolution in the context of host adaptation, plant pathogenesis, and trophic lifestyle is poorly understood. This study applied bioinformatic and phylogenetic methods to examine gene family evolution in C. henricotiae, C. pseudonaviculata and 22 related fungi in the Nectriaceae that vary in pathogenic and saprobic (apathogenic) lifestyles. RESULTS: A total of 19,750 gene families were identified in the 24 genomes, of which 422 were rapidly evolving. Among the six Calonectria species, C. henricotiae and C. pseudonaviculata were the only species to experience high levels of rapid contraction of pathogenesis-related gene families (89% and 78%, respectively). In contrast, saprobic species Calonectria multiphialidica and C. naviculata, two of the closest known relatives of C. henricotiae and C. pseudonaviculata, showed rapid expansion of pathogenesis-related gene families. CONCLUSIONS: Our results provide novel insight into gene family evolution within C. henricotiae and C. pseudonaviculata and suggest gene family contraction may have contributed to limited host-range expansion of these pathogens within the plant family Buxaceae.

Characterization and Aggressiveness of Take-All Root Rot Pathogens Isolated from Symptomatic Bermudagrass Putting Greens.

Take-all root rot is a disease of ultradwarf bermudagrass putting greens caused by Gaeumannomyces graminis (Gg), Gaeumannomyces sp. (Gx), Gaeumannomyces graminicola (Ggram), Candidacolonium cynodontis (Cc), and Magnaporthiopsis cynodontis (Mc). Many etiological and epidemiological components of this disease remain unknown. Improving pathogen identification and our understanding of the aggressiveness of these pathogens along with growth at different temperatures will advance our knowledge of disease development to optimize management strategies. Take-all root rot pathogens were isolated from symptomatic bermudagrass root and stolon pieces from 16 different golf courses. Isolates of Gg, Gx, Ggram, Cc, and Mc were used to inoculate 'Champion' bermudagrass in an in planta aggressiveness assay. Each pathogen was also evaluated at 10, 15, 20, 25, 30, and 35°C to determine growth temperature optima. Infected plant tissue was used to develop a real-time PCR high-resolution melt assay for pathogen detection. This assay was able to differentiate each pathogen directly from infected plant tissue using a single primer pair. In general, Ggram, Gg, and Gx were the most aggressive while Cc and Mc exhibited moderate aggressiveness. Pathogens were more aggressive when incubated at 30°C compared with 20°C. While they grew optimally between 24.4 and 27.8°C, pathogens exhibited limited growth at 35°C and no growth at 10°C. These data provide important information on this disease and its causal agents that may improve take-all root rot management.

"Jumping Jack": Genomic Microsatellites Underscore the Distinctiveness of Closely Related Pseudoperonospora cubensis and Pseudoperonospora humuli and Provide New Insights Into Their Evolutionary Past.

Downy mildews caused by obligate biotrophic oomycetes result in severe crop losses worldwide. Among these pathogens, Pseudoperonospora cubensis and P. humuli, two closely related oomycetes, adversely affect cucurbits and hop, respectively. Discordant hypotheses concerning their taxonomic relationships have been proposed based on host-pathogen interactions and specificity evidence and gene sequences of a few individuals, but population genetics evidence supporting these scenarios is missing. Furthermore, nuclear and mitochondrial regions of both pathogens have been analyzed using microsatellites and phylogenetically informative molecular markers, but extensive comparative population genetics research has not been done. Here, we genotyped 138 current and historical herbarium specimens of those two taxa using microsatellites (SSRs). Our goals were to assess genetic diversity and spatial distribution, to infer the evolutionary history of P. cubensis and P. humuli, and to visualize genome-scale organizational relationship between both pathogens. High genetic diversity, modest gene flow, and presence of population structure, particularly in P. cubensis, were observed. When tested for cross-amplification, 20 out of 27 P. cubensis-derived gSSRs cross-amplified DNA of P. humuli individuals, but few amplified DNA of downy mildew pathogens from related genera. Collectively, our analyses provided a definite argument for the hypothesis that both pathogens are distinct species, and suggested further speciation in the P. cubensis complex.

Competing sexual-asexual generic names in Agaricomycotina (Basidiomycota) with recommendations for use.

With the change to one scientific name for fungal taxa, generic names typified by species with sexual or asexual morph types are being evaluated to determine which names represent the same genus and thus compete for use. In this paper generic names of the Agaricomycotina (Basidiomycota) were evaluated to determine synonymy based on their type. Forty-seven sets of sexually and asexually typified names were determined to be congeneric and recommendations are made for which generic name to use. In most cases the principle of priority is followed. However, 16 generic names are recommended for use that do not have priority and thus need to be protected: Aleurocystis over Matula; Armillaria over Acurtis and Rhizomorpha; Asterophora over Ugola; Botryobasidium over Acladium, Allescheriella, Alysidium, Haplotrichum, Physospora, and Sporocephalium; Coprinellus over Ozonium; Coprinopsis over Rhacophyllus; Dendrocollybia over Sclerostilbum and Tilachlidiopsis; Diacanthodes over Bornetina; Echinoporia over Echinodia; Neolentinus over Digitellus; Postia over Ptychogaster; Riopa over Sporotrichum; Scytinostroma over Artocreas, Michenera, and Stereofomes; Tulasnella over Hormomyces; Typhula over Sclerotium; and Wolfiporia over Gemmularia and Pachyma. Nine species names are proposed for protection: Botryobasidium aureum, B. conspersum, B. croceum, B. simile, Pellicularia lembosporum (syn. B. lembosporum), Phanerochaete chrysosporium, Polyporus metamorphosus (syn. Riopa metamorphosa), Polyporus mylittae (syn. Laccocephalum mylittae), and Polyporus ptychogaster (syn. Postia ptychogaster). Two families are proposed for protection: Psathyrellaceae and Typhulaceae. Three new species names and 30 new combinations are established, and one lectotype is designated.

Genome sequence of Monilinia vaccinii-corymbosi sheds light on mummy berry disease infection of blueberry and mating type.

Mummy berry disease, caused by the fungal pathogen Monilinia vaccinii-corymbosi (Mvc), is one of the most economically important diseases of blueberries in North America. Mvc is capable of inducing two separate blighting stages during its life cycle. Infected fruits are rendered mummified and unmarketable. Genomic data for this pathogen is lacking, but could be useful in understanding the reproductive biology of Mvc and the mechanisms it deploys to facilitate host infection. In this study, PacBio sequencing and Hi-C interaction data were utilized to create a chromosome-scale reference genome for Mvc. The genome comprises nine chromosomes with a total length of 30 Mb, an N50 length of 4.06 Mb, and an average 413X sequence coverage. A total of 9399 gene models were predicted and annotated, and BUSCO analysis revealed that 98% of 1,438 searched conserved eukaryotic genes were present in the predicted gene set. Potential effectors were identified, and the mating-type (MAT) locus was characterized. Biotrophic effectors allow the pathogen to avoid recognition by the host plant and evade or mitigate host defense responses during the early stages of fruit infection. Following locule colonization, necrotizing effectors promote the mummification of host tissues. Potential biotrophic effectors utilized by Mvc include chorismate mutase for reducing host salicylate and necrotrophic effectors include necrosis-inducing proteins and hydrolytic enzymes for macerating host tissue. The MAT locus sequences indicate the potential for homothallism in the reference genome, but a deletion allele of the MAT locus, characterized in a second isolate, indicates heterothallism. Further research is needed to verify the roles of individual effectors in virulence and to determine the role of the MAT locus in outcrossing and population genotypic diversity.

Population Genomics Trace Clonal Diversification and Intercontinental Migration of an Emerging Fungal Pathogen of Boxwood.

Boxwood blight was first documented in Europe, prior to its recent colonization of North America, where it continues to have significant negative impacts on the ornamental industry. Due to near genetic uniformity in the two sister species of fungal plant pathogens that cause boxwood blight, understanding historical disease emergence and predicting future outbreaks is limited. The goal of this research was to apply population genomics to understand the role of pathogen diversification and migration in disease emergence. Specifically, we tested whether the primary pathogen species Calonectria pseudonaviculata has remained genetically isolated from its European-limited sister species C. henricotiae, while diversifying into clonal lineages that have migrated among continents. Whole-genome sequencing identified 1,608 single-nucleotide polymorphisms (SNPs) in 67 C. pseudonaviculata isolates from four continents and 1,017 SNPs in 13 C. henricotiae isolates from Europe. Interspecific genetic differentiation and an absence of shared polymorphisms indicated lack of gene flow between the sister species. Tests for intraspecific genetic structure in C. pseudonaviculata identified four genetic clusters, three of which corresponded to monophyletic phylogenetic clades. Comparison of evolutionary divergence scenarios among the four genetic clusters using approximate Bayesian computation indicated that the two C. pseudonaviculata genetic clusters currently found in the United States were derived from different sources, one from the first genetic cluster found in Europe and the second from an unidentified population. Evidence for multiple introductions of this pathogen into the United States and intercontinental migration indicates that future introductions are likely to occur and should be considered in plant disease quarantine regulation.

One Clonal Lineage of Calonectria pseudonaviculata Is Primarily Responsible for the Boxwood Blight Epidemic in the United States.

Boxwood blight caused by Calonectria pseudonaviculata and C. henricotiae is destroying cultivated and native boxwood worldwide, with profound negative economic impacts on the horticulture industry. First documented in the United States in 2011, the disease has now occurred in 30 states. Previous research showed that global C. pseudonaviculata populations prior to 2014 had a clonal structure, and only the MAT1-2 idiomorph was observed. In this study, we examined C. pseudonaviculata genetic diversity and population structure in the United States after 2014, following the expansion of the disease across the country over the past 5 years. Two hundred eighteen isolates from 21 states were genotyped by sequencing 11 simple sequence repeat (SSR) loci and by MAT1 idiomorph typing. All isolates presented C. pseudonaviculata-specific alleles, indicating that C. henricotiae is still absent in the U.S. states sampled. The presence of only the MAT1-2 idiomorph and gametic linkage disequilibrium suggests the prevalence of asexual reproduction. The contemporary C. pseudonaviculata population is characterized by a clonal structure and composed of 13 multilocus genotypes (SSR-MLGs) unevenly distributed across the United States. These SSR-MLGs grouped into two clonal lineages (CLs). The predominant lineage CL2 (93% of isolates) is the primary contributor to U.S. disease expansion. The contemporary U.S. C. pseudonaviculata population is not geographically subdivided and not genetically differentiated from the U.S. population prior to 2014, but is significantly differentiated from the main European population, which is largely composed of CL1. Our findings provide insights into the boxwood blight epidemic that are critical for disease management and breeding of resistant boxwood cultivars.

Coccinonectria pachysandricola, Causal Agent of a New Foliar Blight Disease of Sarcococca hookeriana.

Woody plants of the Buxaceae, including species of Buxus, Pachysandra, and Sarcococca, are widely grown evergreen shrubs and groundcovers. Severe leaf spot symptoms were observed on S. hookeriana at the U.S. National Arboretum in Washington, DC, in 2016. Affected plants were growing adjacent to P. terminalis exhibiting Volutella blight symptoms. Fungi isolated from both hosts were identical based on morphology and multilocus phylogenetic analysis and were identified as Coccinonectria pachysandricola (Nectriaceae, Hypocreales), causal agent of Volutella blight of Pachysandra species. Pathogenicity tests established that Co. pachysandricola isolated from both hosts caused disease symptoms on P. terminalis and S. hookeriana, but not on B. sempervirens. Artificial inoculations with Pseudonectria foliicola, causal agent of Volutella blight of B. sempervirens, did not result in disease on P. terminalis or S. hookeriana. Wounding enhanced infection by Co. pachysandricola and Ps. foliicola on all hosts tested but was not required for disease development. Genome assemblies were generated for the Buxaceae pathogens that cause Volutella diseases: Co. pachysandricola, Ps. buxi, and Ps. foliicola; these ranged in size from 25.7 to 28.5 Mb. To our knowledge, this foliar blight of S. hookeriana represents a new disease for this host and is capable of causing considerable damage to infected plants.

Identification and characterization of Septoria steviae as the causal agent of Septoria leaf spot disease of stevia in North Carolina.

Stevia (Stevia rebaudiana) is an emerging perennial crop in the southeastern United States. A Septoria leaf spot disease of stevia was first identified on field plantings in Japan in 1978. The pathogen was named Septoria steviae based on a morphological characterization. In 2015, a species of Septoria with morphological characters of S. steviae was isolated from field and greenhouse-grown stevia plants with leaf spot symptoms in North Carolina. In this study, 12 isolates obtained from diseased stevia plants in 2015 and 2016 were characterized and compared with reference strains of S. steviae. Comparisons were based on conidial and pycnidial morphology and multilocus sequence analysis of actin (ACT), ß-tubulin (BT), calmodulin (CAL), nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2 = ITS), nuc rDNA 28S subunit (28S), RNA polymerase II second largest subunit (RPB2), and translation elongation factor-1α (TEF1). Measurements of conidia and pycnidia from symptomatic field leaves and 12 pure cultures grown on nutrient medium were consistent with those previously reported for ex-type strains of S. steviae. North Carolina strains formed a well-supported monophyletic group with ex-type strains of S. steviae. This study represents the first genetic characterization of S. steviae in the United States and provides an experimental framework to elucidate the genetic diversity and disease ecology of field populations of S. steviae.

Multi-locus DNA sequence analysis, antifungal agent susceptibility, and fungal keratitis outcome in horses from Southeastern United States.

Morphological characterization and multi-locus DNA sequence analysis of fungal isolates obtained from 32 clinical cases of equine fungal keratitis (FK) was performed to identify species and determine associations with antifungal susceptibility, response to therapy and clinical outcome. Two species of Aspergillus (A. flavus and A. fumigatus) and three species of Fusarium (F. falciforme, F. keratoplasticum, and F. proliferatum) were the most common fungi isolated and identified from FK horses. Most (91%) equine FK Fusarium nested within the Fusarium solani species complex (FSSC) with nine genetically diverse strains/lineages, while 83% of equine FK Aspergillus nested within the A. flavus clade with three genetically diverse lineages. Fungal species and evolutionary lineage were not associated with clinical outcome. However, species of equine FK Fusarium were more likely (p = 0.045) to be associated with stromal keratitis. Species of Aspergillus were more susceptible to voriconazole and terbinafine than species of Fusarium, while species of Fusarium were more susceptible to thiabendazole than species of Aspergillus. At the species level, A. fumigatus and A. flavus were more susceptible to voriconazole and terbinafine than F. falciforme. Natamycin susceptibility was higher for F. falciforme and A. fumigatus compared to A. flavus. Furthermore, F. falciforme was more susceptible to thiabendazole than A. flavus and A. fumigatus. These observed associations of antifungal sensitivity to natamycin, terbinafine, and thiabendazole demonstrate the importance of fungal identification to the species rather than genus level. The results of this study suggest that treatment of equine FK with antifungal agents requires accurate fungal species identification.

Thermal sensitivity of Calonectria henricotiae and Calonectria pseudonaviculata conidia and microsclerotia.

Knowledge of the thermal sensitivity of conidia and microsclerotia is useful for developing plant disease management approaches that deploy heat to inactivate infectious vegetative propagules of fungal pathogens. For boxwood blight disease, heat treatment of cuttings that harbor conidia and microsclerotia would provide a useful management tool for suppressing the pathogenic activity of Calonectria pseudonaviculata (present in the United States) and C. henricotiae (a quarantine pathogen not present in the United States). In this study, we investigated the thermal sensitivity of conidia and microsclerotia of the boxwood blight pathogens C. henricotiae and C. pseudonaviculata treated in water at 45, 47.5, 50, 52.5, and 55 C. For conidia, as time of exposure increased at each temperature, the proportion of germinated conidia decreased. The predicted time required to inactivate 90% of C. pseudonaviculata conidia (LD90) decreased as water temperature increased from 45 to 55 C and ranged from 35.4 to 5.6 min, respectively. Inactivation of conidia was dependent on isolate, species of Calonectria, and length of exposure at each temperature tested. Microsclerotia of C. henricotiae and C. pseudonaviculata displayed reduced germination with increasing exposure and higher temperatures of hot water. Microsclerotia of C. henricotiae were significantly more resistant to heat treatment than C. pseudonaviculata at 47.5 and 50 C, whereas microsclerotia of both species were rapidly killed at 55 C.

Fine-Scale Genetic Structure and Reproductive Biology of the Blueberry Pathogen Monilinia vaccinii-corymbosi.

The fungus Monilinia vaccinii-corymbosi, a pathogen of Vaccinium spp., requires asexual and sexual spore production to complete its life cycle. A recent study found population structuring of M. vaccinii-corymbosi over a broad spatial scale in the United States. In this study, we examined fine-scale genetic structuring, temporal dynamics, and reproductive biology within a 125-by-132-m blueberry plot from 2010 to 2012. In total, 395 isolates of M. vaccinii-corymbosi were sampled from infected shoots and fruit to examine their multilocus haplotype (MLH) using microsatellite markers. The MLH of 190 single-ascospore isolates from 21 apothecia was also determined. Little to no genetic differentiation and unrestricted gene flow were detected among four sampled time points and between infected tissue types. Discriminant analysis of principal components suggested genetic structuring within the field, with at least K = 3 genetically distinct clusters maintained over four sampled time points. Single-ascospore progeny from eight apothecia had identical MLH and at least two distinct MLH were detected from 13 apothecia. Tests for linkage disequilibrium suggested that genetically diverse ascospore progeny were the product of recombination. This study supports the idea that the fine-scale dynamics of M. vaccinii-corymbosi may be complex, with genetic structuring, inbreeding, and outcrossing detected in the study area.

Phylogenetic relationships of Rhizoctonia fungi within the Cantharellales.

Phylogenetic relationships of Rhizoctonia fungi within the order Cantharellales were studied using sequence data from portions of the ribosomal DNA cluster regions ITS-LSU, rpb2, tef1, and atp6 for 50 taxa, and public sequence data from the rpb2 locus for 165 taxa. Data sets were analysed individually and combined using Maximum Parsimony, Maximum Likelihood, and Bayesian Phylogenetic Inference methods. All analyses supported the monophyly of the family Ceratobasidiaceae, which comprises the genera Ceratobasidium and Thanatephorus. Multi-locus analysis revealed 10 well-supported monophyletic groups that were consistent with previous separation into anastomosis groups based on hyphal fusion criteria. This analysis coupled with analyses of a larger sample of 165 rpb2 sequences of fungi in the Cantharellales supported a sister relationship between the Botryobasidiaceae and Ceratobasidiaceae and a sister relationship of the Tulasnellaceae with the rest of the Cantharellales. The inclusion of additional sequence data did not clarify incongruences observed in previous studies of Rhizoctonia fungi in the Cantharellales based on analyses of a single or multiple genes. The diversity of ecological and morphological characters associated with these fungi requires further investigation on character evolution for re-evaluating homologous and homoplasious characters.

Transcriptomic changes in the plant pathogenic fungus Rhizoctonia solani AG-3 in response to the antagonistic bacteria Serratia proteamaculans and Serratia plymuthica.

BACKGROUND: Improved understanding of bacterial-fungal interactions in the rhizosphere should assist in the successful application of bacteria as biological control agents against fungal pathogens of plants, providing alternatives to chemicals in sustainable agriculture. Rhizoctonia solani is an important soil-associated fungal pathogen and its chemical treatment is not feasible or economic. The genomes of the plant-associated bacteria Serratia proteamaculans S4 and Serratia plymuthica AS13 have been sequenced, revealing genetic traits that may explain their diverse plant growth promoting activities and antagonistic interactions with R. solani. To understand the functional response of this pathogen to different bacteria and to elucidate whether the molecular mechanisms that the fungus exploits involve general stress or more specific responses, we performed a global transcriptome profiling of R. solani Rhs1AP anastomosis group 3 (AG-3) during interaction with the S4 and AS13 species of Serratia using RNA-seq. RESULTS: Approximately 104,504 million clean 75-100 bp paired-end reads were obtained from three libraries, each in triplicate (AG3-Control, AG3-S4 and AG3-AS13). Transcriptome analysis revealed that approximately 10% of the fungal transcriptome was differentially expressed during challenge with Serratia. The numbers of S4- and AS13-specific differentially expressed genes (DEG) were 866 and 292 respectively, while there were 1035 common DEGs in the two treatment groups. Four hundred and sixty and 242 genes respectively had values of log2 fold-change > 3 and for further analyses this cut-off value was used. Functional classification of DEGs based on Gene Ontology enrichment analysis and on KEGG pathway annotations revealed a general shift in fungal gene expression in which genes related to xenobiotic degradation, toxin and antioxidant production, energy, carbohydrate and lipid metabolism and hyphal rearrangements were subjected to transcriptional regulation. CONCLUSIONS: This RNA-seq profiling generated a novel dataset describing the functional response of the phytopathogen R. solani AG3 to the plant-associated Serratia bacteria S4 and AS13. Most genes were regulated in the same way in the presence of both bacterial isolates, but there were also some strain-specific responses. The findings in this study will be beneficial for further research on biological control and in depth exploration of bacterial-fungal interactions in the rhizosphere.

Characterization and colonization of endomycorrhizal Rhizoctonia fungi in the medicinal herb Anoectochilus formosanus (Orchidaceae).

The medicinal effects and techniques for cultivating Anoectochilus formosanus are well-documented, but little is known about the mycorrhizal fungi associated with A. formosanus. Rhizoctonia (Thanatephorus) anastomosis group 6 (AG-6) was the most common species isolated from fungal pelotons in native A. formosanus and represented 67% of the sample. Rhizoctonia (Ceratobasidium) AG-G, P, and R were also isolated and represent the first occurrence in the Orchidaceae. Isolates of AG-6, AG-R, and AG-P in clade I increased seed germination 44-91% and promoted protocorm growth from phases III to VI compared to asymbiotic treatments and isolates of AG-G in clade II and Tulasnella species in clade III. All isolates in clades I to III formed fungal pelotons in tissue-cultured seedlings of A. formosanus, which exhibited significantly greater growth than nonmycorrhizal seedlings. An analysis of the relative effect of treatment ([Formula: see text]) showed that the low level of colonization ([Formula: see text]) by isolates in clade I resulted in a significant increase in seedling growth compared to isolates in clades II (0.63-0.82) and III (0.63-0.75). There was also a negative correlation (r = -0.8801) with fresh plant weight and fungal colonization. Our results suggest that isolates in clade I may represent an important group associated with native populations of A. formosanus and can vary in their ability to establish a symbiotic association with A. formosanus. The results presented here are potentially useful for advancing research on the medicinal properties, production, and conservation of A. formosanus in diverse ecosystems.

Feasibility of flotation concentration of fungal spores as a method to identify toxigenic mushrooms.

PURPOSE: Mushroom poisoning is a recurring and challenging problem in veterinary medicine. Diagnosis of mushroom exposure in animals is hampered by the lack of rapid diagnostic tests. Our study evaluated the feasibility of using flotation concentration and microscopic evaluation of spores for mushroom identification. Evaluation of this method in living animals exposed to toxigenic mushrooms is limited by ethical constraints; therefore, we relied upon the use of an in vitro model that mimics the oral and gastric phases of digestion. METHODS: In our study, mycologist-identified toxigenic (poisonous) and nontoxigenic fresh mushrooms were collected in North Carolina, USA. In phase 1, quantitative spore recovery rates were determined following magnesium sulfate, modified Sheather's sugar solution, and zinc sulfate flotation (n=16 fungal species). In phase 2, mushrooms (n=40 fungal species) were macerated and digested for up to 2 hours in a salivary and gastric juice simulant. The partially digested material was acid neutralized, filtered, and spores concentrated using zinc sulfate flotation followed by microscopic evaluation of spore morphology. RESULTS: Mean spore recovery rates for the three flotation fluids ranged from 32.5% to 41.0% (P=0.82). Mean (± standard error of the mean) Amanita spp. spore recovery rates were 38.1%±3.4%, 36.9%±8.6%, and 74.5%±1.6% (P=0.0012) for the magnesium sulfate, Sheather's sugar, and zinc sulfate solutions, respectively. Zinc sulfate flotation following in vitro acid digestion (phase 2) yielded spore numbers adequate for microscopic visualization in 97.5% of trials. The most common spore shapes observed were globose, spiked, elliptical, smooth and reticulate. CONCLUSION: Flotation can concentrate mushroom spores; however, false negative results can occur. Spore morphology could not be used to differentiate species of mushroom-forming fungi since the spore shape and surface characteristics seen in the present study were often observed with multiple species of mushroom-forming fungi.