Elucidating the effects of organic vs. conventional cropping practice and rhizobia inoculation on rhizosphere microbial diversity and yield of peanut.

Legumes such as peanut (Arachis hypogea) can fulfill most of their nitrogen requirement by symbiotic association with nitrogen-fixing bacteria, rhizobia. Nutrient availability is largely determined by microbial diversity and activity in the rhizosphere that influences plant health, nutrition, and crop yield, as well as soil quality and soil fertility. However, our understanding of the complex effects of microbial diversity and rhizobia inoculation on crop yields of different peanut cultivars under organic versus conventional farming systems is extremely limited. In this research, we studied the impacts of conventional vs. organic cultivation practices and inoculation with commercial vs. single strain inoculum on peanut yield and soil microbial diversity of five peanut cultivars. The experiment was set up in the field following a split-split-plot design. Our results from the 16 S microbiome sequencing showed considerable variations of microbial composition between the cultivation types and inoculum, indicating a preferential association of microbes to peanut roots with various inoculum and cropping system. Alpha diversity indices (chao1, Shannon diversity, and Simpson index) of soil microbiome were generally higher in plots with organic than conventional inorganic practices. The cultivation type and inoculum explained significant differences among bacterial communities. Taxonomic classification revealed two phyla, TM6 and Firmicutes were significantly represented in inorganic as compared to organic soil, where significant phyla were Armatimonadetes, Gemmatimonadetes, Nitrospirae, Proteobacteria, Verrucomicrobia, and WS3. Yields in the organic cultivation system decreased by 10-93% of the yields in the inorganic cultivation system. Cultivar G06 and T511 consistently showed relative high yields in both organic and inorganic trials. Our results show significant two-way interactions between cultivation type and genotype for most of the trait data collected. Therefore, it is critical for farmers to choose varieties based on their cultivation practices. Our results showed that bacterial structure was more uniform in organic fields and microbial diversity in legumes was reduced in inorganic fields. This research provided guides for farmers and scientists to improve peanut yield while promoting microbial diversity and increasing sustainability.

Integration of Phenotypes in Microbiome Networks for Designing Synthetic Communities: a Study of Mycobiomes in the Grafted Tomato System.

Understanding factors influencing microbial interactions, and designing methods to identify key taxa that are candidates for synthetic communities, or SynComs, are complex challenges for achieving microbiome-based agriculture. Here, we study how grafting and the choice of rootstock influences root-associated fungal communities in a grafted tomato system. We studied three tomato rootstocks (BHN589, RST-04-106, and Maxifort) grafted to a BHN589 scion and profiled the fungal communities in the endosphere and rhizosphere by sequencing the internal transcribed spacer (ITS2). The data provided evidence for a rootstock effect (explaining ~2% of the total captured variation, P < 0.01) on the fungal community. Moreover, the most productive rootstock, Maxifort, supported greater fungal species richness than the other rootstocks or controls. We then constructed a phenotype-operational taxonomic unit (OTU) network analysis (PhONA) using an integrated machine learning and network analysis approach based on fungal OTUs and associated tomato yield as the phenotype. PhONA provides a graphical framework to select a testable and manageable number of OTUs to support microbiome-enhanced agriculture. We identified differentially abundant OTUs specific to each rootstock in both endosphere and rhizosphere compartments. Subsequent analyses using PhONA identified OTUs that were directly associated with tomato fruit yield and others that were indirectly linked to yield through their links to these OTUs. Fungal OTUs that are directly or indirectly linked with tomato yield may represent candidates for synthetic communities to be explored in agricultural systems. IMPORTANCE The realized benefits of microbiome analyses for plant health and disease management are often limited by the lack of methods to select manageable and testable synthetic microbiomes. We evaluated the composition and diversity of root-associated fungal communities from grafted tomatoes. We then constructed a phenotype-OTU network analysis (PhONA) using these linear and network models. By incorporating yield data in the network, PhONA identified OTUs that were directly predictive of tomato yield and others that were indirectly linked to yield through their links to these OTUs. Follow-up functional studies of taxa associated with effective rootstocks, identified using approaches such as PhONA, could support the design of synthetic fungal communities for microbiome-based crop production and disease management. The PhONA framework is flexible for incorporation of other phenotypic data, and the underlying models can readily be generalized to accommodate other microbiome or 'omics data.

GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes.

BACKGROUND: CRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing CRISPR-Cas guide RNAs (gRNAs) for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for challenging projects with (i) non-standard Cas enzymes, (ii) non-model organisms, or (iii) projects that need to design a panel of gRNA for genome-wide screens. FINDINGS: GuideMaker can rapidly design gRNAs for gene targets across the genome using a degenerate protospacer-adjacent motif (PAM) and a genome. The tool applies hierarchical navigable small world graphs to speed up the comparison of guide RNAs and optionally provides on-target and off-target scoring. This allows the user to design effective gRNAs targeting all genes in a typical bacterial genome in ∼1-2 minutes. CONCLUSIONS: GuideMaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.

Exposure to airborne bacteria depends upon vertical stratification and vegetation complexity.

Exposure to biodiverse aerobiomes supports human health, but it is unclear which ecological factors influence exposure. Few studies have investigated near-surface green space aerobiome dynamics, and no studies have reported aerobiome vertical stratification in different urban green spaces. We used columnar sampling and next generation sequencing of the bacterial 16S rRNA gene, combined with geospatial and network analyses to investigate urban green space aerobiome spatio-compositional dynamics. We show a strong effect of habitat on bacterial diversity and network complexity. We observed aerobiome vertical stratification and network complexity that was contingent on habitat type. Tree density, closer proximity, and canopy coverage associated with greater aerobiome alpha diversity. Grassland aerobiomes exhibited greater proportions of putative pathogens compared to scrub, and also stratified vertically. We provide novel insights into the urban ecosystem with potential importance for public health, whereby the possibility of differential aerobiome exposures appears to depend on habitat type and height in the airspace. This has important implications for managing urban landscapes for the regulation of aerobiome exposure.

Rootstocks Shape the Rhizobiome: Rhizosphere and Endosphere Bacterial Communities in the Grafted Tomato System.

Root-associated microbes are critical to plant health and performance, although understanding of the factors that structure these microbial communities and the theory to predict microbial assemblages are still limited. Here, we use a grafted tomato system to study the effects of rootstock genotypes and grafting in endosphere and rhizosphere microbiomes that were evaluated by sequencing 16S rRNA. We compared the microbiomes of nongrafted tomato cultivar BHN589, self-grafted BHN589, and BHN589 grafted to Maxifort or RST-04-106 hybrid rootstocks. Operational taxonomic unit (OTU)-based bacterial diversity was greater in Maxifort compared to the nongrafted control, whereas bacterial diversity in the controls (self-grafted and nongrafted) and the other rootstock (RST-04-106) was similar. Grafting itself did not affect bacterial diversity; diversity in the self-graft was similar to that of the nongraft. Bacterial diversity was higher in the rhizosphere than in the endosphere for all treatments. However, despite the lower overall diversity, there was a greater number of differentially abundant OTUs (DAOTUs) in the endosphere, with the greatest number of DAOTUs associated with Maxifort. In a permutational multivariate analysis of variance (PERMANOVA), there was evidence for an effect of rootstock genotype on bacterial communities. The endosphere-rhizosphere compartment and study site explained a high percentage of the differences among bacterial communities. Further analyses identified OTUs responsive to rootstock genotypes in both the endosphere and rhizosphere. Our findings highlight the effects of rootstocks on bacterial diversity and composition. The influence of rootstock and plant compartment on microbial communities indicates opportunities for the development of designer communities and microbiome-based breeding to improve future crop production.IMPORTANCE Understanding factors that control microbial communities is essential for designing and supporting microbiome-based agriculture. In this study, we used a grafted tomato system to study the effect of rootstock genotypes and grafting on bacterial communities colonizing the endosphere and rhizosphere. To compare the bacterial communities in control treatments (nongrafted and self-grafted plants) with the hybrid rootstocks used by farmers, we evaluated the effect of rootstocks on overall bacterial diversity and composition. These findings indicate the potential for using plant genotype to indirectly select bacterial taxa. In addition, we identify taxa responsive to each rootstock treatment, which may represent candidate taxa useful for biocontrol and in biofertilizers.

Penicillin Trunk Injection Affects Bacterial Community Structure in Citrus Trees.

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), an uncultured α-proteobacterium, is the most destructive disease of citrus trees worldwide. In previous studies, trunk injections of penicillin reduced CLas titers and HLB symptoms in citrus. However, antibiotic effects on the whole plant microbial community, which include effects on taxa that interact with CLas, have not yet been addressed. In this study, we investigated the effects of penicillin injection (0, 1000, and 6000 mg L-1) on rhizospheric and endophytic bacterial communities of grapefruit trees in field and greenhouse experiments through culture-independent high-throughput sequencing. DNA extractions from petioles and roots were subjected to 16S rRNA high-throughput sequencing, and reads were clustered by sequence similarity into operational taxonomic units (OTUs). Principal coordinates analysis based on weighted-UniFrac distances did not reveal differences in bacterial communities among treatments in any of the sample sources. However, pairwise linear discriminant analysis indicated significant differences in relative abundance of some taxa (including CLas) among treatments. Network analysis showed that penicillin produced major changes in root bacterial community structure by affecting interspecific microbial associations. This study provides new knowledge of the effect of antimicrobial treatments on interspecific relationships in citrus microbial communities.

Effect of p53 activity on the sensitivity of human glioblastoma cells to PARP-1 inhibitor in combination with topoisomerase I inhibitor or radiation.

Poly (ADP-Ribose) polymerase-1 (PARP-1) is involved in the DNA repairing system by sensing and signaling the presence of DNA damage. Inhibition of PARP-1 is tested in combination with DNA damaging agents such as topoisomerase I inhibitors or ionizing radiations (RT) for the treatment of glioblastoma (GBM). Disruption of p53, widely prevalent in GBMs, plays a major role in DNA repairing system. The current study investigates whether p53 activity has an effect on the sensitivity of human GBM cells to PARP-1 inhibitors in combination with topoisomerase I inhibitor topotecan (TPT) and/or RT. Human GBM cell lines carrying a different functional status of p53 were treated with PARP-1 inhibitor NU1025, in combination with TPT and/or RT. Cytotoxic effects were examined by analyzing the antiproliferative activity, the cell cycle perturbations, and the DNA damage induced by combined treatments. PARP inhibition enhanced the antiproliferative activity, the cell cycle perturbations and the DNA damage induced by both TPT or RT in GBM cells. These effects were influenced by the p53 activity: cells carrying an active p53 were more sensitive to the combination of PARP inhibitor and RT, while cells carrying an inactive p53 displayed a higher sensitivity to the combination of PARP inhibitor and TPT. Our study suggests that p53 activity influences the differential sensitivity of GBM cells to combined treatments of TPT, RT, and PARP inhibitors. © 2014 International Society for Advancement of Cytometry.

Molecular characterization of coprophilous fungal communities reveals sequences related to root-associated fungal endophytes.

This paper reports the use of molecular methods to characterize the coprophilous fungal communities (CFC) that inhabit the dung of four species of mammalian herbivores at two sites, Sevilleta National Wildlife Refuge (SNWR) in New Mexico and Wind Cave National Park (WCNP) in South Dakota. Results reveal that CFC from domesticated cattle (Bos taurus) at SNWR, and bison (Bison bison) and black-tailed prairie dogs (Cynomys ludovicianus) at WCNP were diverse but dominated primarily by members within eight taxonomic orders, including the rarely cultured and anaerobic order Neocallimastigales. In addition, 7.7% (138 of 1,788) of the sequences obtained from all dung samples were at least 97% similar to root-associated fungal (RAF) sequences previously described from blue grama (Bouteloua gracilis), a common forage grass found throughout North America and growing at both study sites. In contrast, 95.8% (295 of 308) of the sequences and four of the total seven operational taxonomic units obtained from pronghorn antelope (Antilocapra americana) dung belonged to the Pleosporalean order. We hypothesize that some herbivore vectors disperse non-systemic (non-clavicipitaceous) fungal endophytes. These dispersal events, it is argued, are most likely to occur via herbivores that occasionally forage and masticate root tissue, especially in arid regions where aboveground vegetation is sparse. The results of this study suggest that some (possibly many) members of the RAF community can expand their ecological role to include colonizing dung.