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Citrus Huanglongbing (HLB; yellow shoot disease) is associated with an unculturable α-proteobacterium “Candidatus Liberibacter asiaticus” (CLas). HLB was found in southern California in 2012, and the current management strategy is based on suppression of the Asian citrus psyllid (Diaphorina citri) that transmits CLas and removal of confirmed CLas-positive trees. Little is known about Asian citrus psyllid-associated bacteria and citrus-associated bacteria in the HLB system. Such information is important in HLB management, particularly for accurate detection of CLas. Recent advancements in next-generation sequencing technology provide new opportunities to study HLB through genomic DNA sequence analyses (metagenomics). In this study, HLB-related bacteria in Asian citrus psyllid and citrus (represented by leaf midrib tissues) samples from southern California were analyzed. A metagenomic pipeline was developed to serve as a prototype for future bacteriomic research. This pipeline included steps of next-generation sequencing in Illumina platform, de novo assembly of Illumina reads, sequence classification using the Kaiju tool, acquisition of bacterial draft genome sequences, and taxonomic validation and diversity evaluation using average nucleotide identity. The identified bacteria in Asian citrus psyllids and citrus together included Bradyrhizobium, Buchnera, Burkholderia, “Candidatus Profftella armature,” “Candidatus Carsonella ruddii,” CLas, Mesorhizobium, Paraburkholderia, Pseudomonas, and Wolbachia. The whole genome of a CLas strain recently found in San Bernardino County was sequenced and classified into prophage typing group 1 (PTG-1), one of the five known CLas groups in California. Based on sequence similarity, Bradyrhizobium and Mesorhizobium were identified as possible source that could interfere with CLas detection using the 16S rRNA gene-based PCR commonly used for HLB diagnosis, particularly at low or zero CLas titer situation.

CandidatusMicrothrix is one of the most common bulking filamentous microorganisms found in activated sludge wastewater treatment plants (WWTPs) across the globe. One species,Ca.M. parvicella, is frequently observed, but global genus diversity, as well as important aspects of its ecology and physiology, are still unknown. Here, we use the MiDAS ecosystem-specific 16S rRNA gene database in combination with amplicon sequencing of Danish and global WWTPs to investigateCa.Microthrix spp. diversity, distribution, and factors affecting their global presence. Only two species were abundant across the world confirming low diversity of the genus: the dominantCa.M. parvicella and an unknown species typically present along withCa.M. parvicella, although usually in lower abundances. Both species were mostly found in Europe at low-to-moderate temperatures and their growth was favored in municipal WWTPs with advanced process designs. As no isolate is available for the novel species, we propose the name “CandidatusMicrothrix subdominans.” Ten high-quality metagenome-assembled genomes recovered from Danish WWTPs, including 6 representing the novelCa.M. subdominans, demonstrated high genetic similarity between the two species with a likely preference for lipids, a putative capability to reduce nitrate and nitrite, and the potential to store lipids and poly-P.Ca.M. subdominans had a potentially more versatile metabolism including additional sugar transporters, higher oxygen tolerance, and the potential to use carbon monoxide as energy source. Newly designed fluorescencein situhybridization probes revealed similar filamentous morphology for both species. Raman microspectroscopy was used to quantify thein situlevels of intracellular poly-P. Despite the observed similarities in their physiology (both by genomes andin situ), the two species showed different seasonal dynamics in Danish WWTPs through a 13-years survey, possibly indicating occupation of slightly different niches. The genomic information provides the basis for future research intoin situgene expression and regulation, while the new FISH probes provide a useful tool for further characterizationin situ. This study is an important step toward understanding the ecology ofCa.Microthrix in WWTPs, which may eventually lead to optimization of control strategies for its growth in this ecosystem.

Huanglongbing, a highly destructive disease of citrus, is associated with the non-culturable phloem-limited α-proteobacterium “Candidatus Liberibacter asiaticus” (CLas). The distribution patterns of CLas in infected plant are variable and not consistent, which make the CLas detection and characterization more challenging. Here, we performed a systemic analysis of CLas distribution in citrus branches and fruits of 14 cultivars. A significantly high concentration of CLas was detected in fruit pith (dorsal vascular bundle) of 14 citrus cultivars collected at fruit maturity season. A 2-year monitoring assay of CLas population in citrus branches of “Shatangju” mandarin (Citrus reticulata Blanco “Shatangju”) revealed that CLas population already exhibited a high level even before the appearance of visual symptoms in the fruit rind. Quantitative analyses of CLas in serial 1.5-cm segments of fruit piths showed the CLas was unevenly distributed within fruit pith and tended to colonize in the middle or distal (stylar end) regions of pith. The use of CLas-abundant fruit pith for dual RNA-seq generated higher-resolution CLas transcriptome data compared with the leaf samples. CLas genes involved in transport system, flagellar assembly, lipopolysaccharide biosynthesis, virulence, stress response, and cell surface structure, as well as host genes involved in biosynthesis of antimicrobial-associated secondary metabolites, was up-regulated in leaf midribs compared with fruit pith. In addition, CLas infection caused the severe collapse in phloem and callose deposition in the plasmodesmata of fruit pith. The ability of fruit pith to support multiplication of CLas to high levels makes it an ideal host tissue for morphological studies and in planta transcriptome analyses of CLas–host interactions.

Huanglongbing is a highly destructive citrus disease associated with “Candidatus Liberibacter asiaticus” (Las), a phloem−limited and non-culturable bacterium, naturally transmitted by the psyllid Diaphorina citri. Although diverse approaches have been used to understand the molecular mechanisms involved in the pathogen–host interaction, such approaches have focused on already infected and/or symptomatic plants, missing early events in the initial days post-inoculation. This study aimed to identify the time course of Las multiplication and whole-plant colonization immediately following inoculation by infected psyllids feeding for 2 days. Thus, the experimental approach was to track Las titers after psyllid inoculation in new shoots (NS) of Citrus × sinensis (susceptible), Murraya paniculata (partially resistant), and Bergera koenigii (fully resistant). Soon after psyllid removal, Las titers dropped until the 10–12th days in all three species. Following this, Las titers increased exponentially only in C. × sinensis and M. paniculata, indicating active bacterial multiplication. In C. × sinensis, Las reached a stationary phase at ∼5 log Las cells/g of tissue from the 40th day onward, while in M. paniculata, Las increased at a lower rate of up to ∼3 log Las cells/g of tissue between the 40th and 60th days, decreasing gradually thereafter and becoming undetectable from the 160th day onward. In B. koenigii, Las titers decreased from the start and remained undetectable. In C. × sinensis, an average of 2.6 log of Las cells/g of tissue was necessary for Las to move out of 50% of the NS in 23.6 days and to colonize the rest of the plant, causing a successful infection. Conversely, the probability of Las moving out of the NS remained below 50% in M. paniculata and zero in B. koenigii. To our knowledge, this is the first study on Las dynamics and whole-plant colonization during the earliest stages of infection. Identification of critical time-points for either successful multiplication or Las resistance may help to elucidate initial events of Las–host interactions that may be missed due to longer sampling intervals and at later stages of infection.

“Candidatus Nitrosocaldaceae” are globally distributed in neutral or slightly alkaline hot springs and geothermally heated soils. Despite their essential role in the nitrogen cycle in high-temperature ecosystems, they remain poorly understood because they have never been isolated in pure culture, and very few genomes are available. In the present study, a metagenomics approach was employed to obtain “Ca. Nitrosocaldaceae” metagenomic-assembled genomes (MAGs) from hot spring samples collected from India and China. Phylogenomic analysis placed these MAGs within “Ca. Nitrosocaldaceae.” Average nucleotide identity and average amino acid identity analysis suggested the new MAGs represent two novel species of “Candidatus Nitrosocaldus” and a novel genus, herein proposed as “Candidatus Nitrosothermus.” Key genes responsible for chemolithotrophic ammonia oxidation and a thaumarchaeal 3HP/4HB cycle were detected in all MAGs. Furthermore, genes coding for urea degradation were only present in “Ca. Nitrosocaldus,” while biosynthesis of the vitamins, biotin, cobalamin, and riboflavin were detected in almost all MAGs. Comparison of “Ca. Nitrosocaldales/Nitrosocaldaceae” with other AOA revealed 526 specific orthogroups. This included genes related to thermal adaptation (cyclic 2,3-diphosphoglycerate, and S-adenosylmethionine decarboxylase), indicating their importance for life at high temperature. In addition, these MAGs acquired genes from members from archaea (Crenarchaeota) and bacteria (Firmicutes), mainly involved in metabolism and stress responses, which might play a role to allow this group to adapt to thermal habitats.

Recent genome-resolved metagenomic analyses of microbial communities from diverse environments have led to the discovery of many novel lineages that significantly expand the phylogenetic breadth of Archaea. Here, we report the genomic characterization of a new archaeal family based on five metagenome-assembled genomes retrieved from acid mine drainage sediments. Phylogenomic analyses placed these uncultivated archaea at the root of the candidate phylum Parvarchaeota, which expand this lesser-known phylum into two family levels. Genes involved in environmental adaptation and carbohydrate and protein utilization were identified in the ultra-small genomes (estimated size 0.53–0.76 Mb), indicating a survival strategy in this harsh environment (low pH and high heavy metal content). The detection of genes with homology to sulfocyanin suggested a potential involvement in iron cycling. Nevertheless, the absence of the ability to synthesize amino acids and nucleotides implies that these archaea may acquire these biomolecules from the environment or other community members. Applying evolutionary history analysis to Parvarchaeota suggested that members of the two families could broaden their niches by acquiring the potentials of utilizing different substrates. This study expands our knowledge of the diversity, metabolic capacity, and evolutionary history of the Parvarchaeota.

Microbial communities with the ability to convert long-chain fatty acids (LCFA) coupled to sulfate reduction can be important in the removal of these compounds from wastewater. In this work, an enrichment culture, able to oxidize the long-chain fatty acid palmitate (C16:0) coupled to sulfate reduction, was obtained from anaerobic granular sludge. Microscopic analysis of this culture, designated HP culture, revealed that it was mainly composed of one morphotype with a typical collar-like cell wall invagination, a distinct morphological feature of the Desulfomonile genus. 16S rRNA gene amplicon and metagenome-assembled genome (MAG) indeed confirmed that the abundant phylotype in HP culture belong to Desulfomonile genus [ca. 92% 16S rRNA gene sequences closely related to Desulfomonile spp.; and ca. 82% whole genome shotgun (WGS)]. Based on similar cell morphology and average nucleotide identity (ANI) (77%) between the Desulfomonile sp. in HP culture and the type strain Desulfomonile tiedjei strain DCB-1T, we propose a novel species designated as “Candidatus Desulfomonile palmitatoxidans.” This bacterium shares 94.3 and 93.6% 16S rRNA gene identity with Desulfomonile limimaris strain DCB-MT and D. tiedjei strain DCB-1T, respectively. Based on sequence abundance of Desulfomonile-morphotype in HP culture, its predominance in the microscopic observations, and presence of several genes coding for enzymes involved in LCFA degradation, the proposed species “Ca. Desulfomonile palmitatoxidans” most probably plays an important role in palmitate degradation in HP culture. Analysis of the growth of HP culture and D. tiedjei strain DCB-1T with short- (butyrate), medium- (caprylate) and long-chain fatty acids (palmitate, stearate, and oleate) showed that both cultures degraded all fatty acids coupled to sulfate reduction, except oleate that was only utilized by HP culture. In the absence of sulfate, neither HP culture, nor D. tiedjei strain DCB-1T degraded palmitate when incubated with Methanobacterium formicicum as a possible methanogenic syntrophic partner. Unlike D. tiedjei strain DCB-1T, “Ca. Desulfomonile palmitatoxidans” lacks reductive dehalogenase genes in its genome, and HP culture was not able to grow by organohalide respiration. An emended description of the genus Desulfomonile is proposed. Our study reveals an unrecognized LCFA degradation feature of the Desulfomonile genus.