First Report of Bacterial Leaf Spot Disease on Sesame (Sesamum indicum L.) Caused by Pseudomonas syringae pv. sesami in North Carolina.

In July 2022, dark brown to black, angular, water-soaked lesions were observed on sesame leaves (Sesamum indicum L.) in a research plot established to assess yield potential for eight varieties at the North Carolina (NC) Sandhills Research Station (Chavez 2023). Symptoms were indicative of a bacterial leaf spot (BLS). At early flowering stage, leaf spots were present on scattered plants; varieties ES108, SS3301, and ES201 exhibited up to 75% disease prevalence, with lower frequency in ES103, S39, S4302, S3251, and S3276. Symptomatic leaves from 3-4 plants were collected on four different dates from July through September. A section of symptomatic tissue was excised and macerated in sterile deionized water (SDW). A 10 µL aliquot was streaked onto SPA medium (15 g sucrose, 5.0 g proteose peptone, 0.50 g MgSO4 7H2O, 0.25 g K2HPO4, 15 g agar per liter of SDW) and incubated at 28ºC. After 72 h, numerous, smooth, white-cream colored, convex-shaped, colonies were individually isolated. Five randomly selected isolates from the different collection dates, designated as AHP108-AHP111 and AHP116, were genotyped. The 16S rRNA, gyrB, rpoD, and gapA genes were sequenced (Heuer et al. 1997; Hwang et al. 2005) and deposited to NCBI (GenBank Accessions: P213467- PP213470; OQ628040-OQ628042; PP214983-PP214994; and PP255798). These five isolates shared 100% sequence identity for gyrB and rpoD. AHP108-AHP111 shared 100% sequence identity for 16S rRNA and gapA, with 99.7% and 90.8% identity, respectively, for AHP116. A phylogenetic tree was inferred from a maximum-likelihood analysis of concatenated gyrB, rpoD, and gapA sequences of the five isolates and the top 11 hts from a blastn search of the NCBI nucleotide database. Those hits included closely related sequences from Pseudomonas syringae pv. sesami type strains ICMP 763T and ICMP 7459T. Based on this phylogenetic analysis AHP108-AHP111 and AHP116 are P. syringae pv. sesami. Recent genomic analysis suggests this pathovar is part of P. amygdali (Gomila et al. 2017), but an official name change has not been proposed. Each of the five isolates were infiltrated into leaves of sesame varieties ES108, ES103, and S327, consistently resulting in similar symptoms. Thus, strain AHP116, as a representative, was used to fulfill Koch's postulates using five, 30-day-old potted sesame plants (var. S3301). Plants were spray-inoculated with a bacterial suspension of ~108 CFU/ml until runoff; plants were incubated in moist chambers 24 h pre and post inoculation at 28ºC with 80% relative humidity and a 12 h photoperiod. At 13 days post inoculation, symptoms resembling those on plants at the Sandhills Research Stations in 2022 were evident. Reisolated bacteria were confirmed to be AHP116 through 16S rRNA and gyrB amplification and sequencing. No symptoms were observed on the five water-inoculated plants. BLS of sesame has been reported in Asia and is thought to be seedborne (Firdous et al. 2009; Prathuangwong and Yowabutra 1997). To our knowledge, this is the first report of P. syringae pv. sesami causing BLS on sesame in North Carolina. Sesame cultivation in the state increased from approximately 2,000 acres in 2022 to 13,000 acres in 2023 and there is interest in cultivating sesame as a rotational and alternative crop because it requires minimal input costs. Potential outbreaks of BLS in this warm, humid region could negatively affect sesame production, where little is known about the economic impact of the disease.

Liberibacter crescens gen. nov., sp. nov., the first cultured member of the genus Liberibacter.

The Gram-stain-negative, rod-shaped bacterial isolate BT-1(T) is the closest relative to the genus 'Candidatus Liberibacter' cultured to date. BT-1(T) was recovered from the phloem sap of a defoliating mountain papaya in Puerto Rico. The BT-1(T) 16S rRNA gene sequence showed that strain BT-1(T) is most closely related to members of the genus 'Ca. Liberibacter' sharing 94.7% 16S rRNA gene sequence similarity with 'Ca. Liberibacter americanus' and 'Ca. Liberibacter asiaticus'. Additionally, average nucleotide identity, 16S rRNA gene sequences and conserved protein sequences supported inclusion of the previously described species of the genus 'Ca. Liberibacter' in a genus with BT-1(T). The prominent fatty acids of isolate BT-1(T) were C18 : 1ω7c (77.2%), C16 : 0 OH (4.8%), C18 : 0 (4.4%) and C16 : 0 (3.5%). Both physiological and genomic characteristics support the creation of the genus Liberibacter, as well as the novel species Liberibacter crescens gen. nov., sp. nov. with type strain BT-1(T) ( = ATCC BAA-2481(T) = DSM 26877(T)).

Interactions between specific phytoplankton and bacteria affect lake bacterial community succession.

Time-series observations and a phytoplankton manipulation experiment were combined to test the hypothesis that phytoplankton succession effects changes in bacterial community composition. Three humic lakes were sampled weekly May-August and correlations between relative abundances of specific phytoplankton and bacterial operational taxonomic units (OTUs) in each time series were determined. To experimentally characterize the influence of phytoplankton, bacteria from each lake were incubated with phytoplankton from one of the three lakes or no phytoplankton. Following incubation, variation in bacterial community composition explained by phytoplankton treatment increased 65%, while the variation explained by bacterial source decreased 64%. Free-living bacteria explained, on average, over 60% of the difference between phytoplankton and corresponding no-phytoplankton control treatments. Fourteen out of the 101 bacterial OTUs that exhibited positively correlated patterns of abundance with specific algal populations in time-series observations were enriched in mesocosms following incubation with phytoplankton, and one out of 59 negatively correlated bacterial OTUs was depleted in phytoplankton treatments. Bacterial genera enriched in mesocosms containing specific phytoplankton assemblages included Limnohabitans (clade betI-A), Bdellovibrio and Mitsuaria. These results suggest that effects of phytoplankton on certain bacterial populations, including bacteria tracking seasonal changes in algal-derived organic matter, result in correlations between algal and bacterial community dynamics.

TWO APPLICATIONS OF PERMUTATION TESTS IN BIOSTASTICS.

We show two examples of how we answer biological questions by converting them into statistical hypothesis testing problems. We consider gene abundance data, and apply permutation tests. Though these tests are simple, they allow us to test biologically relevant hypotheses. Here we present the analysis of data rising from two studies on Type 1 Diabetes. In the first study [3] are interested in comparing the gut bacterial biodiversity in children at risk and not at risk of developing diabetes. In the second study, [4] compare the gut bacterial biodiversity of children in six different sites in USA and Europe. The statistical analyses presented here are parts of the "statistical methods" in two papers mentioned above. Here we offer a detailed explanation of the "Statistical Methods" addressed to readers with a statistics background.

Complete Genome Sequence of Xanthomonas arboricola pv. pruni Strain Xcp1 Isolated in 1984 from a Bacterial Spot Spring Canker on Prunus persica var. nucipersica cv. "Redgold".

Xanthomonas arboricola pv. pruni is an important plant pathogen and the causal agent of bacterial spot of stone fruits (Prunus spp). Here, we report a complete genome of X. arboricola pv. pruni strain Xcp1 generated from hybrid PacBio Sequel and Illumina NextSeq2000 sequencing.

Characterization of ciliate diversity in bromeliad tank waters from the Brazilian Atlantic Forest.

Bromeliads are a diverse group of plants that includes many species whose individuals are capable of retaining water, forming habitats called phytotelmata. These habitats harbor a diversity of organisms including prokaryotes, unicellular eukaryotes, metazoans, and fungi. Among single-celled eukaryotic organisms, ciliates are generally the most abundant. In the present study, we used Illumina DNA sequencing to survey the eukaryotic communities, especially ciliates, inhabiting the tanks of the bromeliads Aechmea gamosepala and Vriesea platynema in the Atlantic Forest of southern Brazil. Filtered sequences were clustered into distinct OTUs using a 99% identity threshold, and then assigned to phylum and genus using a BLAST-based approach (implemented in QIIME) and the SILVA reference database. Both bromeliad species harbored very diverse eukaryotic communities, with Arthropoda and Ciliophora showing the highest abundance (as estimated by the number of sequence reads). The ciliate genus Tetrahymena was the most abundant among single-celled organisms, followed by apicomplexan gregarines and the ciliate genus Glaucoma. Another interesting finding was the presence and high abundance of Trypanosoma in these bromeliad tanks, demonstrating their occurrence in this type of environment. The results presented here demonstrate a hidden diversity of eukaryotes in bromeliad tank waters, opening up new avenues for their in-depth characterization.

Integrating DNA Methylation and Gene Expression Data in the Development of the Soybean-Bradyrhizobium N2-Fixing Symbiosis.

Very little is known about the role of epigenetics in the differentiation of a bacterium from the free-living to the symbiotic state. Here genome-wide analysis of DNA methylation changes between these states is described using the model of symbiosis between soybean and its root nodule-forming, nitrogen-fixing symbiont, Bradyrhizobium diazoefficiens. PacBio resequencing of the B. diazoefficiens genome from both states revealed 43,061 sites recognized by five motifs with the potential to be methylated genome-wide. Of those sites, 3276 changed methylation states in 2921 genes or 35.5% of all genes in the genome. Over 10% of the methylation changes occurred within the symbiosis island that comprises 7.4% of the genome. The CCTTGAG motif was methylated only during symbiosis with 1361 adenosines methylated among the 1700 possible sites. Another 89 genes within the symbiotic island and 768 genes throughout the genome were found to have methylation and significant expression changes during symbiotic development. Of those, nine known symbiosis genes involved in all phases of symbiotic development including early infection events, nodule development, and nitrogenase production. These associations between methylation and expression changes in many B. diazoefficiens genes suggest an important role of the epigenome in bacterial differentiation to the symbiotic state.

Corrigendum: Integrating DNA Methylation and Gene Expression Data in the Development of the Soybean-Bradyrhizobium N2-Fixing Symbiosis.

[This corrects the article on p. 518 in vol. 7, PMID: 27148207.].

Early childhood gut microbiomes show strong geographic differences among subjects at high risk for type 1 diabetes.

OBJECTIVE: Gut microbiome dysbiosis is associated with numerous diseases, including type 1 diabetes. This pilot study determines how geographical location affects the microbiome of infants at high risk for type 1 diabetes in a population of homogenous HLA class II genotypes. RESEARCH DESIGN AND METHODS: High-throughput 16S rRNA sequencing was performed on stool samples collected from 90 high-risk, nonautoimmune infants participating in The Environmental Determinants of Diabetes in the Young (TEDDY) study in the U.S., Germany, Sweden, and Finland. RESULTS: Study site-specific patterns of gut colonization share characteristics across continents. Finland and Colorado have a significantly lower bacterial diversity, while Sweden and Washington state are dominated by Bifidobacterium in early life. Bacterial community diversity over time is significantly different by geographical location. CONCLUSIONS: The microbiome of high-risk infants is associated with geographical location. Future studies aiming to identify the microbiome disease phenotype need to carefully consider the geographical origin of subjects.

Comparative genomics of cultured and uncultured strains suggests genes essential for free-living growth of Liberibacter.

The full genomes of two uncultured plant pathogenic Liberibacter, Ca. Liberibacter asiaticus and Ca. Liberibacter solanacearum, are publicly available. Recently, the larger genome of a closely related cultured strain, Liberibacter crescens BT-1, was described. To gain insights into our current inability to culture most Liberibacter, a comparative genomics analysis was done based on the RAST, KEGG, and manual annotations of these three organisms. In addition, pathogenicity genes were examined in all three bacteria. Key deficiencies were identified in Ca. L. asiaticus and Ca. L. solanacearum that might suggest why these organisms have not yet been cultured. Over 100 genes involved in amino acid and vitamin synthesis were annotated exclusively in L. crescens BT-1. However, none of these deficiencies are limiting in the rich media used to date. Other genes exclusive to L. crescens BT-1 include those involved in cell division, the stringent response regulatory pathway, and multiple two component regulatory systems. These results indicate that L. crescens is capable of growth under a much wider range of conditions than the uncultured Liberibacter strains. No outstanding differences were noted in pathogenicity-associated systems, suggesting that L. crescens BT-1 may be a plant pathogen on an as yet unidentified host.

Compromised gut microbiota networks in children with anti-islet cell autoimmunity.

The gut microbiome is suggested to play a role in the pathogenesis of autoimmune disorders such as type 1 diabetes. Evidence of anti-islet cell autoimmunity in type 1 diabetes appears in the first years of life; however, little is known regarding the establishment of the gut microbiome in early infancy. Here, we sought to determine whether differences were present in early composition of the gut microbiome in children in whom anti-islet cell autoimmunity developed. We investigated the microbiome of 298 stool samples prospectively taken up to age 3 years from 22 case children in whom anti-islet cell autoantibodies developed, and 22 matched control children who remained islet cell autoantibody-negative in follow-up. The microbiome changed markedly during the first year of life, and was further affected by breast-feeding, food introduction, and birth delivery mode. No differences between anti-islet cell autoantibody-positive and -negative children were found in bacterial diversity, microbial composition, or single-genus abundances. However, substantial alterations in microbial interaction networks were observed at age 0.5 and 2 years in the children in whom anti-islet cell autoantibodies developed. The findings underscore a role of the microbiome in the pathogenesis of anti-islet cell autoimmunity and type 1 diabetes.

Ca. Nitrososphaera and Bradyrhizobium are inversely correlated and related to agricultural practices in long-term field experiments.

Agricultural land management, such as fertilization, liming, and tillage affects soil properties, including pH, organic matter content, nitrification rates, and the microbial community. Three different study sites were used to identify microorganisms that correlate with agricultural land use and to determine which factors regulate the relative abundance of the microbial signatures of the agricultural land-use. The three sites included in this study are the Broadbalk Experiment at Rothamsted Research, UK, the Everglades Agricultural Area, Florida, USA, and the Kellogg Biological Station, Michigan, USA. The effects of agricultural management on the abundance and diversity of bacteria and archaea were determined using high throughput, barcoded 16S rRNA sequencing. In addition, the relative abundance of these organisms was correlated with soil features. Two groups of microorganisms involved in nitrogen cycle were highly correlated with land use at all three sites. The ammonia oxidizing-archaea, dominated by Ca. Nitrososphaera, were positively correlated with agriculture while a ubiquitous group of soil bacteria closely related to the diazotrophic symbiont, Bradyrhizobium, was negatively correlated with agricultural management. Analysis of successional plots showed that the abundance of ammonia oxidizing-archaea declined and the abundance of bradyrhizobia increased with time away from agriculture. This observation suggests that the effect of agriculture on the relative abundance of these genera is reversible. Soil pH and NH3 concentrations were positively correlated with archaeal abundance but negatively correlated with the abundance of Bradyrhizobium. The high correlations of Ca. Nitrososphaera and Bradyrhizobium abundances with agricultural management at three long-term experiments with different edaphoclimatic conditions allowed us to suggest these two genera as signature microorganisms for agricultural land use.

Characterization of the Relative Abundance of the Citrus Pathogen Ca. Liberibacter Asiaticus in the Microbiome of Its Insect Vector, Diaphorina citri, using High Throughput 16S rRNA Sequencing.

The relationship between the causal agent of Huanglongbing (HLB), Ca. Liberibacter asiaticus(Las), and the naturally occurring endosymbiotic community of its insect vector, the Asian citrus psyllid (ACP), Diaphorina citri, was studied. Variation was observed in the titer of Las within an ACP population feeding on the same material. The cause of this disparity is unknown, and has implications for Las transmission and the spread of HLB. This study utilizes culture independent methods to establish the relationship between the ACP's microbial community and Las acquisition. DNA from 21 psyllids was amplified using universal 16S rRNA primers with Illumina adaptor regions and a sample-specific 7- base identifier. These amplicons were then batch-sequenced on the Illumina platform. The resulting sequences were separated by the identifier, and compared to known sequences in a 16S rRNA database. The microbial communities of each psyllid were compared to determine whether a correlation exists between the ACP's endosymbionts and level of Las acquisition.ACPs were dominated by the same four bacterialgenera regardless of the abundance of Ca.Liberibacter. A combination of qPCR and Illumina sequencing was used to establish an infection gradient among the sampled ACPs. The Ca. Liberibacter titer within the insect was found to have a strong negative relationship with an endosymbiont residing in the syncytium of the mycetocyte and a positive relationship with Wolbachia. These correlations have implications in the acquisition of Las by the ACP as well as the activities of Las within this vector.

Complete genome sequence of Liberibacter crescens BT-1.

Liberibacter crescens BT-1, a Gram-negative, rod-shaped bacterial isolate, was previously recovered from mountain papaya to gain insight on Huanglongbing (HLB) and Zebra Chip (ZC) diseases. The genome of BT-1 was sequenced at the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida. A finished assembly and annotation yielded one chromosome with a length of 1,504,659 bp and a G+C content of 35.4%. Comparison to other species in the Liberibacter genus, L. crescens has many more genes in thiamine and essential amino acid biosynthesis. This likely explains why L. crescens BT-1 is culturable while the known Liberibacter strains have not yet been cultured. Similar to CandidatusL. asiaticus psy62, the L. crescens BT-1 genome contains two prophage regions.