Publications

4481

A Gram-stain-negative, non-motile, rod-shaped and imazamox-resistant bacterium, designated as H34T, was isolated from soil obtained from a farmland. Strain H34T grew at pH 6.0–9.0 (optimum, 7.0), temperature 15–40 °C (optimum, 30 °C) and salinity of 0–3.0% NaCl (optimum 0.5% NaCl, w/v). Phylogenetic analysis of the 16S rRNA gene and genome sequences showed that strain H34T was affiliated to the family Chitinophagaceae but formed a distinct phyletic branch. The values of 16S rRNA gene sequence similarity, average nt identity (ANI), digital DNA–DNA hybridization (dDDH) and average aa identity (AAI) between strain H34T and closely related type strains of the genera Flavisolibacter, Paraflavisolibacter, Paracnuella, Flaviaesturariibacter and Cnuella were 90.9–92.6%, 68.4–72.1%, 18.7–20.5% and 60.5–65.9%, respectively. The genome size was 7.01 Mb with a G+C content of 56.5 mol%. The major fatty acids (≥10%) were iso-C15 : 0, iso-C15 : 1 G and C16 : 1  ω5c. The only isoprenoid quinone was menaquinone-7. The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified aminophosphoglycolipid, two unidentified phospholipids, three unidentified aminolipids and four unidentified lipids. The distinct phylogenetic relationships; low values of 16S rRNA gene sequence similarity, AAI, ANI and dDDH; and the differences in phenotypic and genotypic characteristics support that strain H34T represents a novel species in a novel genus in the family Chitinophagaceae, for which the name Huijunlia imazamoxiresistens gen. nov., sp. nov. is proposed (type strain H34T=MCCC 1K09117T=KCTC 102287T).

ABSTRACT Huanglongbing (HLB) is a bacterial disease that affects citrus trees and is considered the most severe citrus disease in the world. The three HLB-associated ‘Ca.Liberibacter’ species harbor prophage regions which have been described to play critical roles in their evolution and biology. In this study, we assembled and characterized the accessory genome of the first circularde novo‘CandidatusLiberibacter asiaticus’ (CLas) assembly (V1R1) from Réunion, one of the sparse areas worldwide hosting CLas and ‘Ca.Liberibacter africanus’ (CLaf). This 1,272 Mb-long whole-genome harbored 1,129 coding sequences and two complete prophages, including a 37,934 bp-long Type 1 prophage, frequently present in CLas genomes, and a 40,501 bp-long undescribed CLas prophage designated as P-V1R1-5. Comparative genomic approaches suggested that P-V1R1-5 have all the genetic elements to produce new viral particles and revealed that it likely belongs to a new CLas Type 5 prophage. While being reported for the first time in a CLas strain, P-V1R1-5-like prophages were previously identified in CLaf genomes, making it the first evidence of shared prophage-like sequence among HLB-associated ‘Ca, Liberibacter’ species. Using PCR amplifications targeting P-V1R1-5, we also showed that 85.7% of the tested CLas strains from Réunion and all tested CLaf strains from Madagascar and Réunion harbored a Type 5-like prophage. The identification of CLas Type 5-like prophages not only expanded our knowledge of CLas genomic diversity in Réunion, but also provided new insights into the role of horizontally transferred elements in the evolution of the sympatric HLB-associated bacteria. IMPORTANCE Huanglongbing (HLB) is the most severe citrus disease worldwide. The disease is associated with three ‘CandidatusLiberibacter’ species harboring prophage regions that have been described to play critical roles in their biology. In this study, we assembled and characterized the accessory genome of the first circularde novo‘Ca.Liberibacter asiaticus’ (CLas) assembly from Réunion, one of the very few areas in the world where both of CLas and ‘Ca.Liberibacter africanus’ (CLaf) coexist. Comparative genomic approaches demonstrated that this genome harbored two complete prophages, including a new CLas Type 5 prophage that was previously identified in CLaf but was reported for the first time in a CLas strain. This first evidence of shared prophage-like sequences among HLB-associated species expands our knowledge of CLas genomic diversity, but also provides new insights into the role of the accessory genome in the evolution of these bacteria.

Abstract Immunity is generally considered critical for plant health against pathogen infection. Citrus Huanglongbing (HLB) caused by the phloem colonizing bacterial pathogenCandidatusLiberibacter asiaticus (CLas) was suggested to be a pathogen triggered chronic immune disease. However, the genetic evidence and mechanistic understanding for such a disease model is lacking. Here, we show CLas triggers phloem cell death, reactive oxygen species (ROS) production and callose deposition in photosynthesis tissues, but little or none in non-photosynthesis tissues of citrus. We further demonstrate that CLas triggers ROS production in chloroplasts. Overexpression offlavodoxin(Fld), an electron shuttle, in chloroplasts reduced ROS production, cell death and HLB symptoms, but not phloem callose deposition induced by CLas. Knockout, silencing, and overexpression of phloem callose synthase genesCsCalS7a,CsCalS7b, orCsCalS7cdemonstrated that phloem callose deposition also caused phloem cell death. Trunk injection with 2-deoxy-d-glucose, a callose deposition inhibitor, reduced fruit drop and increased fruit yield of HLB symptomatic trees in field trials. Using tomato-Ca. L. psyllaurous (Lpsy) as a model, knockout ofEds1andPad4but notRbohB,Bik1andSobir1abolished ROS production, phloem callose deposition, cell death and disease damages caused by Lpsy. This study provides genetic evidence forCa. Liberibacter-triggered immune disease and reveals that tuning plant immune responses convertsCa. Liberibacter into a benign endophyte, providing a promising strategy for precision breeding to enhance resistance/tolerance against HLB.

A Gram-positive, oval-shaped and non-motile strain, designated MMS21-DH1MA12T, was isolated from riverside soil and subjected to polyphasic taxonomic characterization. Strain MMS21-DH1MA12T grew optimally in tryptic soy agar, forming yellow-coloured, convex and smooth colonies. The optimal growth conditions of the strain were 30 °C, pH 7 and absence of NaCl. Strain MMS21-DH1MA12T shared the highest 16S rRNA gene sequence similarity with Agrococcus sediminis NS18T (97.3%), and then with Diaminobutyricimonas aerilata 6408 J-67T (97.1%) and Mycetocola miduiensis CGMCC 1.11101 T (96.9%), all belonging to the family Microbacteriaceae. Strain MMS21-DH1MA12T had a genome size of 2.4 Mbp and G+C content of 62.6%. The digital DNA–DNA hybridization (dDDH), orthologous average nucleotide identity (orthoANI) and average amino acid identity (AAI) values between strain MMS21-DH1MA12T and related type strains of the genera within the family Microbacteriaceae were no higher than 22.2%, 71.54% and 60.19%, respectively. The dDDH, orthoANI and AAI values of MMS21-DH1MA12T with related taxa were no higher than 22.2%, 71.54% and 61.96%, thus showing a distant relationship of the strain with other members of Microbacteriaceae. It was also notable that the species sharing the highest similarities were different for all three indices. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. The main cellular fatty acids were anteiso-C15 : 1, antesio-C17 : 1 and iso-C15 : 0. The predominant menaquinones were MK-10 and MK-11. The combination of physiological and biochemical properties, as well as chemotaxonomic features, clearly separated strain MMS21-DH1MA12T from related taxa of Microbacteriaceae. It is, thus, evident that MMS21-DH1MA12T represents a novel species in a novel genus, for which the name Humidisolicoccus flavus gen. nov., sp. nov. is proposed (type strain=MMS21-DH1MA12T = KCTC 49751T = LMG 32525T=JCM 37009T).

‘Candidatus Phytoplasma mali’ is associated with apple proliferation, a devastating disease in fruit production. Using genome analysis, a gene encoding a hemolysin-like protein was identified. It was postulated that this protein could be an effector. However, the function of this protein is unknown. It is shown that the hemolysin-like protein binds to a GTP binding protein, Toc33, of Arabidopsis thaliana in yeast two-hybrid analysis and that the Toc33-binding domain is located in the C-terminus of the domain of unknown function (DUF21) of the protein. The biochemical studies reveal that the protein can hydrolyze phosphate of purine and pyrimidine nucleotides. Transgenic Nicotiana benthamiana plants expressing the protein show no discernible change in phenotype. Phytoplasma have a much-reduced genome, lacking important genes for catabolic pathways or nucleotide production; therefore, the hemolysin-like protein plays a role in the uptake of plant nucleotides from their host and hydrolyzes these nucleotides for energy and their own biosynthesis.

The order Sphingomonadales strains are globally distributed in various biomes and are renowned for their biodegradable and biosynthesis capabilities. At present, it consists of 4 families and 49 genera making it the third largest order within the class Alphaproteobacteria. However, their taxonomy remains complex, especially due to polyphyly in the family Sphingomonadaceae. In this study, we collected 429 Sphingomonadales type strain genomes, reconstructed robust phylogenomic relationships, and proposed delineation thresholds at the genus and family levels based on average amino acid identities (AAI) and evolutionary distances (ED). Based on the maximum-likelihood and Bayesian phylogenomic trees reconstructed by two molecular sets determined by orthologous sequence identity and the Genome Taxonomy Database, the consensus degree values were all higher than 90%, revealing that those phylogenomic trees had similar topological structures. By confirming monophyletic taxa and determining stable nodes, we reclassified the order Sphingomonadales into thirteen families including nine novel ones. AAI calculations indicated that the average intra-family AAI values ranged from 0.62 to 0.84, while inter-family ones were 0.51 to 0.60. ED summaries demonstrated that the average and median intra-family ED values were 0.16 to 0.57, and inter-family ones ranged from 0.50 to 1.22. Comparisons of AAI and ED values calculated by using genomic and phylogenetic analyses supported that those 13 families were significantly separated with p values < 2.2×10−16. Thus, it was speculated that the AAI and ED thresholds for distinguishing different families were <0.6 and >0.5, respectively. Additionally, we reclassified 163 species into new genera with their phylogenetic topologies, according to the previous genus AAI and ED boundaries of 0.7 and 0.4. Our study is the first genomic-based study of the order Sphingomonadales and will promote further insights into the evolution of this order.

Following Rule 51b(4) of the International Code of Nomenclature of Prokaryotes (2022 Revision) (ICNP), the prokaryotic generic name Moorella Collins et al. 1994 is illegitimate, as it is a later homonym of the fungal genus name Moorella P. Rag. Rao & D. Rao 1964 (Ascomycota: Pezizomycotina). Accordingly, as required by Rule 54 of the ICNP, we propose the replacement name Neomoorella and the replacement type species name Neomoorella thermoacetica. We also propose replacement names for the six other species with validly published and not rejected names belonging to the genus Moorella, for one species with effectively but not validly published name and for the family name Moorellaceae and the order name Moorellales.

A Gram-stain-negative coccobacillus, LMG 32992T, was isolated from water that had collected in a tyre in Pingtung, Donggang Township, Taiwan. Upon preliminary 16S rRNA gene sequence analysis, it was most closely related to members of the genus Ralstonia (16S rRNA gene sequence similarities of 96.7–97.5%). The present study aimed to elucidate its taxonomic position and to propose a formal classification. To this end, the complete genome sequence was determined, and taxonomic, phylogenomic, metabolic and physiological analyses were performed. Comparative genomic analyses demonstrated that strain LMG 32992T and another unclassified strain, Burkholderiaceae bacterium PBA, which was isolated earlier from textile wastewater in Malaysia, represented a single novel species within a novel genus of the family Burkholderiaceae. The G+C content of the LMG 32992T genomic DNA was 63.77 mol%. Genomic analyses and growth tests demonstrated that LMG 32992T had an asaccharolytic metabolism but that it was well-equipped to synthetize, if necessary autotrophically, and transform all required carbohydrates and that it used the Krebs and related cycles to generate reductive power for a heterotrophic energy metabolism. We propose the name Imbroritus primus gen. nov., sp. nov. with strain LMG 32992T (=CIP 112179T=BCRC 81361T=A30B1T) as the type strain, for this novel taxon.

Huanglongbing (citrus greening disease) is caused by the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) (Alphaproteobacteria) and is one of the most destructive bacterial-vector diseases affecting the citrus industry. The bacterium is transmitted by the Asian citrus psyllid (ACP; Diaphorina citri). Early detection in citrus trees is challenging due to uneven distribution of CLas throughout the tree and a long pre-symptomatic phase of the disease. Due to these limitations, ACP sampling has been suggested as a more reliable early detection strategy. The objective of this study was to develop and optimize approaches for detecting CLas in ACP adults and nymphs collected in citrus groves in California using real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). The goal was to establish the optimal number of ACP adults and nymphal instar life stages (stages 1–2, 3, or 4–5) that yielded the most reliable detection of CLas (Cq values ≤ 38). Results indicated that CLas detection correlated with psyllid developmental stage, with the 4th–5th instar nymphs (sample size of five to ten per tube) or adult ACP (sample size of three to ten per tube) providing the most consistent qPCR detection. While CLas detection rates increased with adult ACP age, nymphs were preferred for field sampling as adult ACP might have dispersed from non-infected trees, potentially misrepresenting the grove’s CLas status. Detection by droplet digital PCR confirmed the presence and genome copies of CLas in a subset of ACP across life stages. In field populations, detection rates in nymphs were consistent or stable throughout the year, whereas CLas detection in adults exhibited seasonal variation, with CLas detection and genome load peaking in January. These targeted ACP sampling strategies and optimized laboratory processing methods will facilitate CLas detection in psyllids for streamlining citrus greening disease management.

Jujube witches’ broom (JWB) disease, caused by Candidatus Phytoplasma ziziphi (Ca. P. ziziphi), severely threatens the production of Chinese jujube (Ziziphus jujuba Mill.). Emerging evidence highlights the critical role of phytoplasma-secreted effectors in pathogenesis, though few have been functionally characterized. Here, we identified a Sec-dependent effector, JWB790, from Ca. P. ziziphi, which was shown to suppress plant immunity. Through transient expression assays in Nicotiana benthamiana, pathogen inoculation assays, the generation of transgenic Arabidopsis thaliana plants, and RNA-seq-based transcriptomic profiling, we systematically investigated the virulence function of JWB790. Our findings revealed that JWB790 is highly expressed in JWB-infected tissues. The transient expression of JWB790 in N. benthamiana suppressed BAX-induced cell death and H2O2 accumulation. Furthermore, the stable overexpression of JWB790 in A. thaliana compromised disease resistance, accompanied by reduced H2O2 accumulation and callose deposition triggered by flg22. Additionally, the RNA-seq analysis of JWB790 transgenic Arabidopsis plants indicated that the overexpression of JWB790 altered the expression of biotic stress-related genes. In summary, JWB790 is a virulence factor that suppresses plant immunity and promotes pathogen proliferation. These results advance our understanding of Ca. P. ziziphi pathogenesis.