Microbiology (medical)

“Candidatus Liberibacter asiaticus” (CLas) is an unculturable phloem-limited α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). HLB is currently threatening citrus production worldwide. Understanding the CLas biology is critical for HLB management. In this study, a novel single-stranded DNA (ssDNA) phage, CLasMV1, was identified in a CLas strain GDHZ11 from Guangdong Province of China through a metagenomic analysis. The CLasMV1 phage had a circular genome of 8,869 bp with eight open reading frames (ORFs). While six ORFs remain uncharacterized, ORF6 encoded a replication initiation protein (RIP), and ORF8 encoded a major capsid protein (MCP). Based on BLASTp search against GenBank database, amino acid sequences of both MCP and RIP shared similarities (coverage > 50% and identity > 25%) to those of phages in Microviridae, an ssDNA phage family. Phylogenetic analysis revealed that CLasMV1 MCP and RIP sequences were clustered with genes from CLas and “Ca. L. solanacearum” (CLso) genomes and formed a unique phylogenetic lineage, designated as a new subfamily Libervirinae, distinct to other members in Microviridae family. No complete integration form but partial sequence (∼1.9 kb) of CLasMV1 was found in the chromosome of strain GDHZ11. Read-mapping analyses on additional 15 HiSeq data sets of CLas strains showed that eight strains harbored complete CLasMV1 sequence with variations in single-nucleotide polymorphisms (SNPs) and small sequence insertions/deletions (In/Dels). PCR tests using CLasMV1-specific primer sets detected CLasMV1 in 577 out of 1,006 CLas strains (57%) from southern China. This is the first report of Microviridae phage associated with CLas, which expands our understanding of phage diversity in CLas and facilitates current research in HLB.

Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas) brings a great concern about the phloem nutrient transport in diseased plants. There is an urgent need to find the best management strategies to reduce the losses in the citrus industry worldwide. Endophytic bacteria are negatively affected by CLas pathogen, and these endophytes are associated with improved availability of nutrients and pathogen resistance. This study underpins the relationship between CLas pathogen, endophyte population and nutrients availability in citrus plants. The citrus plants were treated with Bacillus subtilis L1-21 and Hoagland solution to find out synergism efficacy to mitigate citrus HLB. We showed that citrus shoots in the presence of 50% Hoagland solution displayed maximum number of endophytes with 6.28 × 103 to 3.04 × 105 CFU/g. Among 50 candidate strains, B. subtilis L1-21 emerged as potential antagonist against surrogate strain Xanthomonas citri subsp. citri. The citrus half-leaf method identified that application of endophyte L1-21 with 50% Hoagland solution successfully reduces the CLas abundance. We point out that this combination results in a higher number of endophytes population with 2.52 × 104 to 9.11 × 106 CFU/g after 60 days, and reduces CLas pathogen abundance in asymptomatic HLB plants. In HLB symptomatic citrus plants, B. subtilis L1-21 potentially increases the endophyte population from 1.11 × 104 to 5.26 × 107 CFU/g in the presence of Hoagland solution, and pathogen abundance was reduced from 9.51 × 105 to 1.06 × 104 copies/g. Altogether, we suggested that the presence of endophyte L1-21 with Hoagland solution is more effective in HLB asymptomatic citrus plants, but a slight reduction of pathogen was observed in symptomatic plants. The findings revealed the role of indigenous citrus endophyte B. subtilis L1-21 along with other nutrients in the reduction of CLas pathogen abundance inside symptomatic and asymptomatic plants in citrus endophyte–nutrient–pathogen interplay.

The potential metabolism and ecological roles of many microbial taxa remain unknown because insufficient genomic data are available to assess their functional potential. Two such microbial “dark matter” taxa are the Candidatus bacterial phyla Cloacimonadota and Omnitrophota, both of which have been identified in global anoxic environments, including (but not limited to) organic-carbon-rich lakes. Using 24 metagenome-assembled genomes (MAGs) obtained from an Antarctic lake (Ace Lake, Vestfold Hills), novel lineages and novel metabolic traits were identified for both phyla. The Cloacimonadota MAGs exhibited a capacity for carbon fixation using the reverse tricarboxylic acid cycle driven by oxidation of hydrogen and sulfur. Certain Cloacimonadota MAGs encoded proteins that possess dockerin and cohesin domains, which is consistent with the assembly of extracellular cellulosome-like structures that are used for degradation of polypeptides and polysaccharides. The Omnitrophota MAGs represented phylogenetically diverse taxa that were predicted to possess a strong biosynthetic capacity for amino acids, nucleosides, fatty acids, and essential cofactors. All of the Omnitrophota were inferred to be obligate fermentative heterotrophs that utilize a relatively narrow range of organic compounds, have an incomplete tricarboxylic acid cycle, and possess a single hydrogenase gene important for achieving redox balance in the cell. We reason that both Cloacimonadota and Omnitrophota form metabolic interactions with hydrogen-consuming partners (methanogens and Desulfobacterota, respectively) and, therefore, occupy specific niches in Ace Lake.

Sap-feeding insects in the order Hemiptera associate with obligate endosymbionts that are required for survival and facultative endosymbionts that can potentially modify resistance to stress, enemies, development, and reproduction. In the superfamily Psylloidea, the jumping plant lice (psyllids), less is known about the diversity and prevalence of their endosymbionts compared to other sap-feeding pests such as aphids (Aphididae). To address this knowledge gap, using 16S rRNA sequencing we identify symbionts across divergent psyllid host lineages from around the world. Taking advantage of a new comprehensive phylogenomic analyses of Psylloidea, we included psyllid samples from 44 species of 35 genera of five families, collected from 11 international locations for this study. Across psyllid lineages, a total of 91 OTUs were recovered, predominantly of the Enterobacteriaceae (68%). The diversity of endosymbionts harbored by each psyllid species was low with an average of approximately 3 OTUs. Two clades of endosymbionts (clade 1 and 2), belonging to Enterobacteriaceae, were identified that appear to be long term endosymbionts of the psyllid families Triozidae and Psyllidae, respectively. We also conducted high throughput metagenomic sequencing on three Ca. Liberibacter infected psyllid species (Russelliana capsici, Trichochermes walkeri, and Macrohomotoma gladiata), initially identified from 16S rRNA sequencing, to obtain more genomic information on these putative Liberibacter plant pathogens. The phylogenomic analyses from these data identified a new Ca. Liberibacter species, Candidatus Liberibacter capsica, that is a potential pathogen of solanaceous crops. This new species shares a distant ancestor with Ca. L. americanus, which occurs in the same range as R. capsici in South America. We also detected the first association between a psyllid specializing on woody hosts and the Liberibacter species Ca. L. psyllaurous, which is a globally distributed pathogen of herbaceous crop hosts in the Solanaceae. Finally, we detected a potential association between a psyllid pest of figs (M. gladiata) and a Ca. Liberibacter related to Ca. L. asiaticus, which causes severe disease in citrus. Our findings reveal a wider diversity of associations between facultative symbionts and psyllids than previously reported and suggest numerous avenues for future work to clarify novel associations of ecological, evolutionary, and pathogenic interest.

Rubbery taproot disease of sugar beet (RTD), associated with ‘Candidatus Phytoplasma solani’, appeared in 2020 on an epidemic scale in northern Serbia and southern Slovakia, situated at opposite edges of the Pannonian Plain. In the affected locations where the disease was assessed, symptomatic sugar beets were analysed for phytoplasma infection. Additionally, multilocus sequence analyses of ‘Ca. P. solani’ strains on epidemiologically informative marker genes (tuf, stamp and vmp1) were performed. Symptomatic sugar beets from other countries of the Pannonian Plain (Croatia, Hungary and Austria), one sample from Germany, and red beets from Serbia were included in the analyses. ‘Ca. P. solani’ was detected in sugar beet in all assessed countries, as well as in red beet. Molecular analyses revealed the high genetic variability of ‘Ca. P. solani’ with the presence of all four tuf-types (a, b1, b2 and d), 14 stamp genotypes (seven new) and five vmp1 profiles (one new). The most common multilocus genotype in Serbia, Slovakia, Croatia, and Hungary was dSTOLg (tuf-d/STOL/V2-TA). It was dominant on sites with epidemic RTD outbreaks in the Pannonian Plain and in several sugar beet fields with non-epidemic RTD occurrence suggesting the prevalence of a particular epidemiological pathway during the epidemic’s phases.

SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors. Although SAP11 of different phytoplasma species share similar activities, their protein sequences differ greatly. Here, we demonstrate that the SAP11-like protein of ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) strain PM19 localizes into the plant nucleus without requiring the anticipated nuclear localization sequence (NLS). We show that the protein induces crinkled leaves and siliques, and witches’ broom symptoms, in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to six members of class I and all members of class II TCP transcription factors of A. thaliana in yeast two-hybrid assays. We also identified a 17 amino acid stretch previously predicted to be a nuclear localization sequence that is important for the binding of some of the TCPs, which results in a crinkled leaf and silique phenotype in transgenic A. thaliana. Moreover, we provide evidence that the SAP11-like protein has a destabilizing effect on some TCPs in vivo.

Nitrogen cycling microbes, including nitrite-oxidizing bacteria (NOB), perform critical ecosystem functions that help mitigate anthropogenic stresses and maintain ecosystem health. Activity of these beneficial nitrogen cycling microbes is dictated in part by the microorganisms’ response to physicochemical conditions, such as temperature, pH, and nutrient availability. NOB from the newly described Candidatus Nitrotoga genus have been detected in a wide range of habitats across the globe, yet only a few organisms within the genus have been physiologically characterized. For freshwater systems where NOB are critical for supporting aquatic life, Ca. Nitrotoga have been previously detected but little is known about the physiological potential of these organisms or their response to changing environmental conditions. Here, we determined functional response to environmental change for a representative freshwater species of Ca. Nitrotoga (Ca. Nitrotoga sp. CP45, enriched from a Colorado river). The physiological findings demonstrated that CP45 maintained nitrite oxidation at pH levels of 5–8, at temperatures from 4 to 28°C, and when incubated in the dark. Light exposure and elevated temperature (30°C) completely halted nitrite oxidation. Ca. Nitrotoga sp. CP45 maintained nitrite oxidation upon exposure to four different antibiotics, and potential rates of nitrite oxidation by river sediment communities were also resilient to antibiotic stress. We explored the Ca. Nitrotoga sp. CP45 genome to make predictions about adaptations to enable survival under specific conditions. Overall, these results contribute to our understanding of the versatility of a representative freshwater Ca. Nitrotoga sp. Identifying the specific environmental conditions that maximize NOB metabolic rates may ultimately direct future management decisions aimed at restoring impacted systems.

Boreal lakes and ponds produce two-thirds of the total natural methane emissions above the latitude of 50° North. These lake emissions are regulated by methanotrophs which can oxidize up to 99% of the methane produced in the sediments and the water column. Despite their importance, the diversity and distribution of the methanotrophs in lakes are still poorly understood. Here, we used shotgun metagenomic data to explore the diversity and distribution of methanotrophs in 40 oxygen-stratified water bodies in boreal and subarctic areas in Europe and North America. In our data, gammaproteobacterial methanotrophs (order Methylococcales) generally dominated the methanotrophic communities throughout the water columns. A recently discovered lineage of Methylococcales, Candidatus Methylumidiphilus, was present in all the studied water bodies and dominated the methanotrophic community in lakes with a high relative abundance of methanotrophs. Alphaproteobacterial methanotrophs were the second most abundant group of methanotrophs. In the top layer of the lakes, characterized by low CH4 concentration, their abundance could surpass that of the gammaproteobacterial methanotrophs. These results support the theory that the alphaproteobacterial methanotrophs have a high affinity for CH4 and can be considered stress-tolerant strategists. In contrast, the gammaproteobacterial methanotrophs are competitive strategists. In addition, relative abundances of anaerobic methanotrophs, Candidatus Methanoperedenaceae and Candidatus Methylomirabilis, were strongly correlated, suggesting possible co-metabolism. Our data also suggest that these anaerobic methanotrophs could be active even in the oxic layers. In non-metric multidimensional scaling, alpha- and gammaproteobacterial methanotrophs formed separate clusters based on their abundances in the samples, except for the gammaproteobacterial Candidatus Methylumidiphilus, which was separated from these two clusters. This may reflect similarities in the niche and environmental requirements of the different genera within alpha- and gammaproteobacterial methanotrophs. Our study confirms the importance of O2 and CH4 in shaping the methanotrophic communities and suggests that one variable cannot explain the diversity and distribution of the methanotrophs across lakes. Instead, we suggest that the diversity and distribution of freshwater methanotrophs are regulated by lake-specific factors.