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Neonicotinoids, a widely used class of insecticides, enter surface waters through stormwater runoff and threaten the health of aquatic ecosystems. Imidacloprid is a commonly used neonicotinoid in the United States and is present in surface waters across North America. White-rot wood decay fungi can degrade neonicotinoid compounds, and their ability to grow on woody materials makes them well suited to passive fungal bioremediation applications. However, there is limited research on the viability of fungal bioremediation for stormwater treatment. In this study, we investigated the rate of imidacloprid degradation by the wood decay fungus Phanerodontia chrysosporiumin suspended and attached growth systems. Cytochrome P450 enzyme inhibitor 1-aminobenzotriazole was used to investigate the degradation mechanism. Imidacloprid and fungal biomass were measured over four weeks in a synthetic stormwater fungal batch system and transformation products were identified. More than 90% of imidacloprid was degraded in four weeks in liquid and wood chip experiments, with more than 50% of degradation occurring in the first week. The cytochrome P450 enzyme inhibitor had no effect on imidacloprid degradation, suggesting that these enzymes were not responsible for the degradation under the imposed nutrient-limited conditions. Estimated first-order reaction rate constants for the liquid and wood chip culture treatments were 0.17 d−1and 0.11 d−1respectively. We tentatively identified three previously described transformation products, imidacloprid-nitrosoguanidine, imidacloprid-olefin, and 6-chloro-3-pyridylmethanol in fungal-amended samples. To our knowledge, this research represents the first study to examine and demonstrate fungal degradation of imidacloprid in synthetic stormwater, and the first to explore this degradation with wood chips as a fungal growth medium. Significant degradation (>50%) of imidacloprid after 7 days by P. chrysosporiumsuggests that fungal-based biodegradation may provide a valuable stormwater treatment option.

ABSTRACTNatural history collections contain billions of specimens which are used to inform our understanding of the biodiversity of our planet. Digitized natural history collections (dNHCs) provide an accessible resource for multiple audiences, including K-12 teachers. The incorporation of dNHCs in K-12 classrooms provides an opportunity to engage students in authentic and practical science experiences. More importantly, through collaboration with museum educators the use of dNHCs can address some of the challenges K-12 teachers face when addressing the Next Generation Science Standards. Using primary data sources will ensure students understand the natural world through the perspective of scientific practices that involve data analyses, structured experiential and inquiry-based learning, and constructing explanations in a hypothesis-testing framework.

ABSTRACTKnowledge 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. henricotiaeand C. pseudonaviculatatreated 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. pseudonaviculataconidia (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. henricotiaeand C. pseudonaviculatadisplayed reduced germination with increasing exposure and higher temperatures of hot water. Microsclerotia of C. henricotiaewere significantly more resistant to heat treatment than C. pseudonaviculataat 47.5 and 50 C, whereas microsclerotia of both species were rapidly killed at 55 C.

The soil fungus Rhizoctonia solaniproduces phytotoxic phenylacetic acid (PAA) and hydroxy (OH-) and methoxy (MeO-) derivatives of PAA. However, limited information is available on the specific role that these compounds play in the development of Rhizoctoniadisease symptoms and concentration(s) required to induce a host response. Reports that PAA inhibits the growth of R. solaniconflict with the established ability of the fungus to produce and metabolize PAA. Experiments were conducted to clarify the role of the PAA metabolic complex in Rhizoctoniadisease. In this study the concentration of PAA and derivatives required to induce tomato root necrosis and stem canker, in the absence of the fungus, and the concentration that inhibits mycelial growth of R. solaniwere determined. The effect of exogenous PAA and derivatives of PAA on tomato seedling growth also was investigated. Growth of tomato seedlings in medium containing 0.1–7.5 mM PAA and derivatives induced necrosis of up to 85% of root system. Canker development resulted from injection of tomato seedling stems with 7.5 mM PAA, 3-OH-PAA, or 3-MeO-PAA. PAA in the growth medium reduced R. solanibiomass, with 50% reduction observed at 7.5 mM. PAA, and derivatives were quantified from the culture medium of 14 isolates of R. solanibelonging to three distinct anastomosis groups by GC-MS. The quantities ranged from below the limit of detection to 678 nM, below the concentrations experimentally determined to be phytotoxic. Correlation analyses revealed that isolates of R. solanithat produced high PAA and derivatives in vitro also caused high mortality on tomato seedlings. The results of this investigation add to the body of evidence that the PAA metabolic complex is involved in Rhizoctoniadisease development but do not indicate that production of these compounds is the primary or the only determinant of pathogenicity.

Eighteen isolates from sugar beet roots associated with an unknown etiology were characterized based on observations of morphological characters, hyphal growth at 4–28 C, production of phenol oxidases and sequence analysis of internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA (rDNA). The isolates did not produce asexual or sexual spores, had binucleate hyphal cells with clamp connections, grew 4–22 C with estimated optimal growth at 14.5 C and formed a dark brown pigment on potato dextrose or malt extract agar amended with 0.5% tannic acid. Color changes observed when solutions of gum guiac, guiacol and syringaldzine were applied directly to mycelium grown on these media indicated that all isolates produced phenol oxidases. Sequences of ITS and LSU regions on the rDNA gene from 15 isolates were 99.2–100% identical, and analysis of sequence data with maximum likelihood and maximum parsimony suggest that the isolates from sugar beet roots are phylogenetically related to Athelia bombacina, Granulobasidium vellereumand Cyphella digitalis. High statistical support for both loci under different criteria confirmed that Athelia bombacinawas consistently the closest known relative to the sugar beet isolates. Additional taxonomic investigations are needed before species can be clarified and designated for these isolates.

Isolates from closely related fungi in the Rhizoctoniaspecies complex were examined for the occurrence of the M2 double-stranded RNA (dsRNA) by amplifying a conserved 1000 nucleotide region of the dsRNA with reverse transcription PCR. The M2 dsRNA was detected in representative isolates belonging to three anastomosis groups (AG) of R. solani(AG-1-IA, AG-4 and AG-6; teleomorph = Thanatephorus) and four AGs of binucleate Rhizoctonia(AGA, AG-F, AG-R and AG-U; teleomorph = Ceratobasidium). Amplified PCR products from the 3′ region of the M2 dsRNA from a representative sample of 12 isolates from eight different AGs were sequenced and subjected to parsimony analysis and coalescent simulations to infer ancestral lineages and to reconstruct the ancestral history of haplotypes. Seven dsRNA haplotypes were inferred from the sample of 12 isolates. One haplotype was composed of only isolates of Ceratobasidiumbelonging to different AGs. The rooted gene genealogies from coalescent simulations suggested that the ancestral M2 dsRNA haplotype most likely evolved in Thanatephorus(anamorph = R. solaniAG-1-IA) and has been acquired recently by isolates of Ceratobasidium. Reconstruction of the ancestral history of haplotypes with a parsimony-based approach that assumes both mutation and recombination suggested that four haplotypes recombined before coalescing to their most recent common ancestor, while three haplotypes coalesced without recombination in the recent past. There was no unique association of haplotype within a specific AG of either Ceratobasidiumor Thanatephorusto support co-evolution of the M2 dsRNA within the fungal host. To our knowledge this is the first report of a dsRNA occurring in Ceratobasidiumthat also is present in Thanatephorus.

Horizontal transmission of the 3.57 kb M2 double-stranded RNA (dsRNA) between mycelia of somatically incompatible isolates of Rhizoctonia solanianastomosis group 3 (AG-3), an economically important pathogen of cultivated plants in the family Solanaceae, was investigated. Nine donor isolates of R. solaniAG-3 containing the M2 dsRNA were paired on potato-dextrose agar with each of three different recipient isolates where the M2 dsRNA was absent. Reverse-transcription PCR (RT-PCR) was used to detect horizontal transmission of the M2 dsRNA via hyphal anastomosis from donor to recipient isolates by examining hyphal explants taken 3 cm from the hyphal interaction zone. PCR-RFLP genetic-based markers of two nuclear loci and one mitochondrial locus were used to confirm identity and transmission between donor and recipient isolates of R. solaniAG-3. The frequency of transmission observed between 72 pairings of the eight donor and three recipient isolates was approximately 4% of the total pairings, and differences in the phenotype of the recipient isolates after acquisition of the M2 dsRNA via horizontal transmission were observed. To our knowledge this represents the first demonstration of transmission of dsRNA between genetically different individuals of R. solaniconfirmed with nuclear and mitochondrial markers. These results suggest that transmission can occur between somatically incompatible isolates of R. solaniAG-3 but that maintenance of the dsRNA in the recipient isolates was not stable after repeated subculturing on nutrient medium.

Leaf, stem, and tuber samples collected from diseased potato plants exhibiting tospovirus-like symptoms in eastern North Carolina (NC) were assayed for the presence ofTomato spotted wilt virus(TSWV) by serology (ImmunoStrips) and reverse transcription-polymerase chain reaction (RT-PCR) methods. All symptomatic samples tested positive for TSWV with serology and RT-PCR. Mechanical inoculation of the indicator hostNicotiana benthamianawith symptomatic leaf, stem, and tuber tissue resulted in plants with symptoms of systemic virus infection. The presence of TSWV in infected tissue ofN. benthamianawas confirmed with serology, RT-PCR, and sequence analysis of the nucleocapsid (N) protein gene. Phylogenetic analysis of the predicted N protein of three isolates of TSWV from potato in NC and 11 isolates obtained from peanut, pepper, and tomato in Georgia and NC suggested that the isolates were related. One isolate of TSWV from potato in NC was selected for vector transmission tests, and the virus was transmitted to potato with tobacco thrips (Frankliniella fusca), but no vertical transmission from infected seed tubers to actively developing potato sprouts, stems or leaves was observed. This is the first documentation of the occurrence, isolation and characterization of TSWV from field-grown potato in North America.

Anastomosis group 3 (AG-3) of Rhizoctonia solani(teleomorph = Thanatephorus cucumeris) is frequently associated with diseases of potato (AG-3 PT) and tobacco (AG-3 TB). Although isolates of R. solaniAG-3 from these two Solanaceous hosts are somatically related based on anastomosis reaction and taxonomically related based on fatty acid, isozyme and DNA characters, considerable differences are evident in their biology, ecology, and epidemiology. However, genetic diversity among field populations of R. solaniAG-3 PT and TB has not been documented. In this study, the genetic diversity of field populations of R. solaniAG-3 PT and AG-3 TB in North Carolina was examined using somatic compatibility and amplified fragment length polymorphism (AFLP) criteria. A sample of 32 isolates from potato and 36 isolates from tobacco were paired in all possible combinations on PDA plus activated charcoal and examined for their resulting somatic interactions. Twenty-eight and eight distinct somatic compatibility groups (SCG) were identified in the AG-3 PT and AG-3 TB samples, respectively. AFLP analyses indicated that each of the 32 AG-3 PT isolates had a distinct AFLP phenotype, whereas 28 AFLP phenotypes were found among the 36 isolates of AG-3 TB. None of the AG-3 PT isolates were somatically compatible or shared a common AFLP phenotype with any AG-3 TB isolate. Clones (i.e., cases where two or more isolates were somatically compatible and shared the same AFLP phenotype) were identified only in the AG-3 TB population. Four clones from tobacco represented 22% of the total population. All eight SCG from tobacco were associated with more than one AFLP phenotype. Compatible somatic interactions between AG-3 PT isolates occurred only between certain isolates from the same field (two isolates in each of four different fields), and when this occurred AFLP phenotypes were similar but not identical.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to identify and differentiate genotypes of Rhizoctonia solanianastomosis group 3 subgroup PT (AG-3 PT), a fungal pathogen of potato. Polymorphic co-dominant single-locus PCR-RFLP markers were identified after sequencing of clones from a genomic library and digestion with restriction enzymes. Multilocus genotypes were determined by a combination of PCR product and digestion with a specific restriction enzyme for each of seven loci. A sample of 104 isolates from one commercial field in each of five counties in eastern North Carolina was analyzed, and evidence for high levels of gene flow between populations was revealed. When data were clone-corrected and samples pooled into one single North Carolina population, random associations of alleles were found for all loci or pairs of loci, indicating random mating. However, when all genotypes were analyzed, the observed genotypic diversity deviated from panmixia and alleles within and between loci were not randomly associated. These findings support a model of population structure for R. solaniAG-3 PT on potato that includes both recombination and clonality.

AbstractThe phylogenetic relationships of anastomosis groups (AG) of Rhizoctoniaassociated with Ceratobasidiumand Thanatephorusteleomorphs were determined by cladistic analyses of internal transcribed spacer (ITS) and 28S large subunit (LSU) regions of nuclear-encoded ribosomal DNA (rDNA). Combined analyses of ITS and LSU rDNA sequences from 41 isolates representing 28 AG of Ceratobasidiumand Thanatephorussupported at least 12 monophyletic groupings within Ceratobasidiumand Thanatephorus.There was strong support for separation of Ceratobasidiumand Thanatephorus, however, six sequences representing different AG of Ceratobasidiumgrouped with certain sequences within the Thanatephorusclade. Phylogenetic analysis of ITS sequence data from 122 isolates revealed 31 genetically distinct groups from Thanatephorus(21 groups) and Ceratobasidium(10 groups) that corresponded well with previously recognized AG or AG subgroups. Although phylogenetic analysis of ITS sequences provided evidence that several AG of Ceratobasidiummay be more closely related with some AG from Thanatephorus, these relationships were not as strongly supported by bootstrap analysis.

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: Aleurocystisover Matula; Armillariaover Acurtisand Rhizomorpha; Asterophoraover Ugola; Botryobasidiumover Acladium, Allescheriella, Alysidium, Haplotrichum, Physospora,and Sporocephalium; Coprinellusover Ozonium; Coprinopsisover Rhacophyllus; Dendrocollybiaover Sclerostilbumand Tilachlidiopsis; Diacanthodesover Bornetina; Echinoporiaover Echinodia; Neolentinusover Digitellus; Postiaover Ptychogaster; Riopaover Sporotrichum; Scytinostromaover Artocreas, Michenera, and Stereofomes; Tulasnellaover Hormomyces; Typhulaover Sclerotium;and Wolfiporiaover Gemmulariaand 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: Psathyrellaceaeand Typhulaceae. Three new species names and 30 new combinations are established, and one lectotype is designated.

Micropeltopsis cannabis sp. nov. and Orbilia luteola (Roum.) comb. nov. are proposed. New Cannabis host associations include binucleate Rhizoctonia spp., Curvularia cymbopogonis, Sphaerotheca macularis, Glomus mosseae, and Pestalotiopsis sp. The geographic ranges of Pseudoperonspora cannabina, Septoria neocannabina and Fusarium graminearum are expanded.