Seasonal Distribution of Cyanobacteria in Three Urban Eutrophic Lakes Results from an Epidemic-like Response to Environmental Conditions.

Cyanobacterial communities of three co-located eutrophic sandpit lakes were surveyed during 2016 and 2017 over season and depth using high-throughput DNA sequencing of the 16S rRNA gene. All three lakes were stratified except during April 2017 when the lakes were recovering from a strong mixing event. 16S rRNA gene V4 sequences were parsed into operational taxonomic units (OTUs) at 99% sequence identity. After rarefaction of 139 samples to 25,000 sequences per sample, a combined total of 921,529 partial 16S rRNA gene sequences were identified as cyanobacteria. They were binned into 19,588 unique cyanobacterial OTUs. Of these OTUs, 11,303 were Cyanobium. Filamentous Planktothrix contributed 1537 and colonial Microcystis contributed 265. The remaining 6482 OTUs were considered unclassified. For Planktothrix and Microcystis one OTU accounted for greater than 95% of the total sequences for each genus. However, in both cases the non-dominant OTUs clustered with the dominant OTUs by date, lake, and depth. All Planktothrix OTUs and a single Cyanobium OTU were detected below the oxycline. All other Cyanobium and Microcystis OTUs were detected above the oxycline. The distribution of Cyanobium OTUs between lakes and seasons can be explained by an epidemic-like response where individual OTUs clonally rise from a diverse hypolimnion population when conditions are appropriate. The importance of using 99% identity over the more commonly used 97% is discussed with respect to cyanobacterial community structure. The approach described here can provide another valuable tool for assessing cyanobacterial populations and provide greater insight into the controls of cyanobacterial blooms.

Binning Singletons: Mentoring through Networking at ASM Microbe 2019.

The American Society for Microbiology (ASM) national conference, Microbe, is the flagship meeting for microbiologists across the globe. The presence of roughly 10,000 attendees provides enormous opportunities for networking and learning. However, such a large meeting can be intimidating to many, especially early career scientists, students, those attending alone, and those from historically underrepresented groups. While mentorship is widely valued by ASM and its members, finding concrete ways to develop new and diverse mentoring opportunities can be a challenge. We recognized the need for an initiative aimed at expanding peer-to-peer mentoring, facilitating networking, and providing support for Microbe attendees; therefore, we created the program Binning Singletons for ASM Microbe 2019. The program consisted of five steps named after tools or phenomena in the profession of microbiology: (i) Identify the Singletons (e.g., individuals attending alone), (ii) Bin the Singletons, (iii) Horizontal Transfer, (iv) Quorum Sensing, and (v) Exponential Growth. These steps resulted in the matching of participants unsure of how to get the most out of their conference experience (e.g., singletons) with mentors who assisted with meeting planning, networking, and/or impostor syndrome. Started on social media only a month before ASM Microbe 2019, the program successfully launched despite limited time and resources. Binning Singletons improved inclusivity and networking opportunities for participants at the conference. Here, we discuss what worked, and what can be improved, with an eye toward development of the Binning Singletons model for future conferences to provide opportunities to increase inclusivity, networking, and accessibility for singletons and build a stronger scientific community.

Quantifying stream periphyton assemblage responses to nutrient amendments with a molecular approach.

Nutrient (nitrogen [N] and phosphorus [P]) pollution is a pervasive water quality issue in the USA for small streams and rivers. The effect of nutrients on the biotic condition of streams is often evaluated with biological indicators such as macroinvertebrate assemblages or periphyton assemblages, particularly diatoms. Molecular approaches facilitate the use of periphyton assemblages as bioindicators because periphyton is diverse and its composition as a whole, rather than just diatoms, soft-bodied algae, or any single group, may convey additional information about responses to nutrients. To further develop the concept that a taxonomically-broad evaluation of periphyton assemblages could be useful for developing stream bioindicators, we examined microbial assemblage composition with both 16S and 18S rRNA genes, enabling us to evaluate composition in 3 domains. We measured otherwise unknown nutrient responses of different periphyton groups in situ with experiments that used glass fiber filters to allow diffusion of amended nutrients into a stream. We deployed these experimental setups in 2 streams that differ in the extent of agricultural land-use in their catchments in the southeastern USA. Experiments consisted of controls, N amendments, P amendments, and both N and P amendments. Periphyton assemblages that grew on the filters differed significantly by stream, date or season, and nutrient treatment. Assemblage differences across treatments were more consistent among Bacteria and Archaea than among eukaryotes. Effects of nutrient amendments were more pronounced in the stream with less agricultural land use and, therefore, lower nutrient loading than in the stream with more agricultural land use and higher nutrient loading. Combined N and P amendments decreased species richness and evenness for Bacteria and Archaea by ∼36 and ∼9%, respectively, compared with controls. Indicator species analysis revealed that specific clades varied in their response to treatments. Indicators based on the responses of these indicator clades were related to nutrient treatments across sites and seasons. Analyses that included all the taxa in a domain did not resolve differences in responses to N vs P. Instead, better resolution was achieved with an analysis focused on diatoms, which responded more strongly to P than N. Overall, our results showed that in situ nutrient-diffusing substrate experiments are a useful approach for describing assemblage responses to nutrients in streams. This type of molecular approach may be useful to environmental agencies and stakeholders responsible for assessing and managing stream water quality and biotic condition.

Dissecting Resistance to Phytophthora cinnamomi in Interspecific Hybrid Chestnut Crosses Using Sequence-Based Genotyping and QTL Mapping.

The soilborne oomycete Phytophthora cinnamomi-which causes root rot, trunk cankers, and stem lesions on an estimated 5,000 plant species worldwide-is a lethal pathogen of American chestnut (Castanea dentata) as well as many other woody plant species. P. cinnamomi is particularly damaging to chestnut and chinquapin trees (Castanea spp.) in the southern portion of its native range in the United States due to relatively mild climatic conditions that are conductive to disease development. Introduction of resistant genotypes is the most practical solution for disease management in forests because treatment with fungicides and eradication of the pathogen are neither practical nor economically feasible in natural ecosystems. Using backcross families derived from crosses of American chestnuts with two resistant Chinese chestnut cultivars Mahogany and Nanking, we constructed linkage maps and identified quantitative trait loci (QTLs) for resistance to P. cinnamomi that had been introgressed from these Chinese chestnut cultivars. In total, 957 plants representing five cohorts of three hybrid crosses were genotyped by sequencing and phenotyped by standardized inoculation and visual examination over a 6-year period from 2011 to 2016. Eight parental linkage maps comprising 7,715 markers were constructed, and 17 QTLs were identified on four linkage groups (LGs): LG_A, LG_C, LG_E, and LG_K. The most consistent QTLs were detected on LG_E in seedlings from crosses with both 'Mahogany' and 'Nanking' and LG_K in seedlings from 'Mahogany' crosses. Two consistent large and medium effect QTLs located ∼10 cM apart were present in the middle and at the lower end of LG_E; other QTLs were considered to have small effects. These results imply that the genetic architecture of resistance to P. cinnamomi in Chinese chestnut × American chestnut hybrid progeny may resemble the P. sojae-soybean pathosystem, with a few dominant QTLs along with quantitatively inherited partial resistance conferred by multiple small-effect QTLs.

Resistance to Phytophthora cinnamomi in American Chestnut (Castanea dentata) Backcross Populations that Descended from Two Chinese Chestnut (Castanea mollissima) Sources of Resistance.

Restoration of American chestnut (Castanea dentata) depends on combining resistance to both the chestnut blight fungus (Cryphonectria parasitica) and Phytophthora cinnamomi, which causes Phytophthora root rot, in a diverse population of C. dentata. Over a 14-year period (2004 to 2017), survival and root health of American chestnut backcross seedlings after inoculation with P. cinnamomi were compared among 28 BC3, 66 BC4, and 389 BC3F3 families that descended from two BC1 trees (Clapper and Graves) with different Chinese chestnut grandparents. The 5% most resistant Graves BC3F3 families survived P. cinnamomi infection at rates of 75 to 100% but had mean root health scores that were intermediate between resistant Chinese chestnut and susceptible American chestnut families. Within Graves BC3F3 families, seedling survival was greater than survival of Graves BC3 and BC4 families and was not genetically correlated with chestnut blight canker severity. Only low to intermediate resistance to P. cinnamomi was detected among backcross descendants from the Clapper tree. Results suggest that major-effect resistance alleles were inherited by descendants from the Graves tree, that intercrossing backcross trees enhances progeny resistance to P. cinnamomi, and that alleles for resistance to P. cinnamomi and C. parasitica are not linked. To combine resistance to both C. parasitica and P. cinnamomi, a diverse Graves backcross population will be screened for resistance to P. cinnamomi, survivors bred with trees selected for resistance to C. parasitica, and progeny selected for resistance to both pathogens will be intercrossed.

Effects of large-scale poultry farms on aquatic microbial communities: a molecular investigation.

The effects of large-scale poultry production operations on water quality and human health are largely unknown. Poultry litter is frequently applied as fertilizer to agricultural lands adjacent to large poultry farms. Run-off from the land introduces a variety of stressors into the surface waters including nutrients, antimicrobials and pathogenic bacteria. The Delaware, Maryland and Virginia (Delmarva) Peninsula has the highest concentration of broiler chickens per farm acre in the United States and provides an ideal location for studying the effects of stressors from poultry farms. We investigated potential effects by characterizing shifts in the structure of aquatic bacterial communities. DNA was isolated from microorganisms in water samples from streams and rivers at varying distances from, or having different frequencies of, litter applications. Fingerprints of 16S rDNA amplicons from bacteria in water samples collected during late summer 2001 to late spring 2002 were produced by denaturing gradient gel electrophoresis (DGGE). A statistical analysis of multiple fingerprints from each sampling location demonstrated that each site harboured a bacterial community significantly different from the communities at other sites. Similarly, the bacterial communities from each sampling time differed significantly from communities at other sampling times. Most importantly, a competitive, library-based analysis showed time of sampling (month) had a greater effect on community structure than did location.

Use of composite data sets for source-tracking enterococci in the water column and shoreline interstitial waters on Pensacola Beach, Florida.

Sources of Enterococcus faecalis isolates from Pensacola Beach, FL. were identified using a library-based approach by applying the statistical method of average similarity to single and composite data sets generated from separate analyses. Data sets included antibiotic resistance analysis (ARA), rep-fingerprints, and fatty acid methyl ester (FAME) profiles. Use of a composite data set composed of ARA and rep-fingerprints, added to the confidence of the identifications. The addition of FAME data to composite data sets did not add to the confidence of identifications. Source identification was performed to better understand risk associated with higher densities of enterococci found in swash zone interstitial water (SZIW) as compared to adjacent bathing water on Pensacola Beach, FL. The "swash zone" is that area of the beach continually washed over by waves. As the potential sources of enterococci were limited in this environment, only two library units, sea gull and human, were constructed. Identification of the beach isolates using a composite data set indicated a sea gull origin. The clonality of the beach isolates suggested that the beach environment selects certain subspecies of E. faecalis.