Publications

3681

AbstractInfection with the bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) is suspected to cause severe damage in carrot leading to high carrot weight loss. This study investigates three main aspects: (i) whether there is a reduction of carrot root weight under field conditions due to Lso infection; (ii) the correlation between Lso infection rate in carrot plants and occurrence of the psyllid Trioza apicalis as the known vector for Lso in carrot, and (iii) the comparison between symptoms described in literature and observed symptoms associated with Lso infection. Therefore, field surveys were conducted from 2018 to 2021 on organically managed carrot fields in Lower Saxony, Germany. Two Rebell orange sticky traps were placed per field replaced on a weekly basis. Captured T. apicalis were morphologically identified and counted. Carrot plant samples were collected from T. apicalis infested fields. Discolouration of foliage and further symptoms on carrots were investigated. For detection of Lso in carrot plants and psyllids, the samples were analysed using PCR. The infection rate of carrot plants varied between 2.5% and 80% per field with low abundance of T. apicalis. Of the asymptomatic plants, 39.4% were infected with Lso, while 80.9% of the symptomatic plants were infected with Lso. The weight of Lso-positive carrot samples was not significantly reduced compared to Lso-negative samples. No economically relevant losses of carrot yield were reported by farmers participating in this study. This indicates that there was no effect of Lso infection on carrot root weight during this study. Of the symptoms associated with a Lso infection in carrots, only leaf discolouration could be confirmed. Overall, the bacterium is present in organically grown carrot plants in Lower Saxony but no major carrot root weight losses were observed.

Two Gram-stain-positive, aerobic, non-spore-forming, non-motile, irregular rod-shaped actinobacteria, designated as D2-41T and D3-21, were isolated from soil samples collected in a natural cave in Jeju, Republic of Korea. Both of the isolates were shown to share 100 % 16S rRNA sequence identity. The cell wall contained meso-diaminopimelic acid, arabinose and galactose. The predominant menaquinone was MK-8(H2). The polar lipids contained phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified aminolipid, an unidentified aminoglycolipid, an unidentified phospholipid and two unidentified lipids. The predominant fatty acids were C16 : 0 and summed feature 3 (C16 : 1  ω7c and/or iso-C15 : 0 2-OH). Mycolic acids of C30–C38 were present. The 16S rRNA gene trees showed that the organisms occupied a distinct position remotely located from recognized genera within the order Mycobacteriales, albeit with the 16S rRNA gene similarities of 97.0–97.1 % with Rhodococcus olei, Rhodococcus rhodnii and Rhodococcus triatomae. The genome sizes and DNA G+C contents of strains D2-41T and D3-21 were 4.77–4.88 Mbp and 69.8 mol%, respectively. Both of the isolates shared an average nucleotide identity of 99.4 % and digital DNA–DNA hybridization of 95.2 % to each other, revealing that strains D2-41T and D3-21 belonged to the same species. In the core genome-based phylogenomic tree, both of the isolates were found to be closely associated with members of the genus Tomitella. However, strains D2-41T and D3-21 revealed the highest amino acid identity values (mean 66.5 %, range 66.2–67.0 % with the genus Prescottella of the family Nocardiaceae, followed by the genus Tomitella (mean 64.1 %, range 63.6–64.7 %) of the family Tomitellaceae. Based on the combined data obtained here, the novel isolates belong to a new genus of the new family for which the name Speluncibacter jeojiensis gen. nov. sp. nov. is proposed, with Speluncibacteraceae fam. nov. The type strain is strain D2-41T (=KACC 17930T=DSM 101875T).

Abstract Phytoplasmas manipulate host plant development to benefit insect vector colonization and their own invasion. However, the virulence factors and mechanisms underlying small-leaf formation caused by jujube witches’ broom (JWB) phytoplasmas remain largely unknown. Here, effectors SJP1 and SJP2 from JWB phytoplasmas were identified to induce small-leaf formation in jujube (Ziziphus jujuba). In vivo interaction and expression assays showed that SJP1 and SJP2 interacted with and stabilized the transcription factor ZjTCP2. Overexpression of SJP1 and SJP2 in jujube induced ZjTCP2 accumulation. In addition, the abundance of miRNA319f_1 was significantly reduced in leaves of SJP1 and SJP2 transgenic jujube plants and showed the opposite pattern to the expression of its target, ZjTCP2, which was consistent with the pattern in diseased leaves. Overexpression of ZjTCP2 in Arabidopsis promoted ectopic leaves arising from the adaxial side of cotyledons and reduced leaf size. Constitutive expression of the miRNA319f_1 precursor in the 35S::ZjTCP2 background reduced the abundance of ZjTCP2 mRNA and reversed the cotyledon and leaf defects in Arabidopsis. Therefore, these observations suggest that effectors SJP1 and SJP2 induced small-leaf formation, at least partly, by interacting with and activating ZjTCP2 expression both at the transcriptional and the protein level, providing new insights into small-leaf formation caused by phytoplasmas in woody plants.

The family Peptostreptococcaceae, which contains 15 genera including Clostridioides, presently lacks proper circumscription. Using 52 available genomes for Peptostreptococcaceae species, we report comprehensive phylogenomic and comparative analyses to reliably discern their evolutionary relationships. In phylogenetic trees based on core genome proteins and 16S rRNA gene sequences, the examined species formed a strongly supported clade designated as Peptostreptococcaceae sensu stricto. This clade encompassed the genera Peptostreptococcus (type genus), Asaccharospora, Clostridioides, Intestinibacter, Paeniclostridium, Paraclostridium, Peptacetobacter, Romboutsia and Terrisporobacter, and two misclassified species (viz. Eubacterium tenue and ‘Clostridium dakarense’). The distinctness of this clade is strongly supported by eight identified conserved signature indels (CSIs), which are specific for the species from this clade. Based on the robust evidence provided by presented studies, we are proposing the emendment of family Peptostreptococcaceae to only the genera within the Peptostreptococcaceae sensu stricto clade. We also report 67 other novel CSIs, which reliably demarcate different Peptostreptococcaceae species clades and clarify the classification of some misclassified species. Based on the consistent evidence obtained from different presented studies, we are making the following proposals to clarify the classification of Peptostreptococcaceae species: (i) transfer of Eubacterium tenue, Paeniclostridium ghonii and Paeniclostridium sordellii as comb. nov. into the genus Paraclostridium; (ii) transfer of Clostridioides mangenotii as a comb. nov. into Metaclostridioides gen. nov.; (iii) classification of ‘Clostridium dakarense’ as a novel species Faecalimicrobium dakarense gen. nov., sp. nov. (type strain FF1T; genome and 16S rRNA accession numbers GCA_000499525.1 and KC517358, respectively); (iv) transfer of two misclassified species, Clostridium paradoxum and Clostridium thermoalcaliphilum, into Alkalithermobacter gen. nov.; and (v) proposals for two novel families, Peptoclostridiaceae fam. nov. and Tepidibacteraceae fam. nov., to accommodate remaining unclassified Peptostreptococcaceae genera. The described CSIs specific for different families and genera provide novel and reliable means for the identification, diagnostics and biochemical studies on these bacteria.

ABSTRACT Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense ( Ca . M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca . M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca . M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca . M. massiliense and possible transfer between hosts. IMPORTANCE Methanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca . M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.

Psyllids, members of the family Triozidae, represent a potential threat to the cultivation of solanaceous and apiaceous crops worldwide, mainly as vectors of the phloem-restricted bacterium ‘Candidatus Liberibacter solanacearum’ (Lso). The Lso haplotypes C, D and E are known to affect apiaceous crops, such as carrot and celery, in several European countries. In Italy, data on the incidence and natural spread of both Lso and psyllids have not been reported so far. In this study, the presence of the vectors was investigated in a main Italian district for carrot production, the “Altopiano del Fucino” area (Central Italy). Both occasional and regular surveys were carried out on a total of five carrot fields and one potato field in 2021 and 2022. Bactericera trigonica (Hodkinson), which is known to efficiently transmit Lso to carrots, was found to be well-established in the area. High levels of population density were recorded in the summer period (more than 100 adult specimens per trap caught every two weeks) and then sharply decreased after the carrot harvest, confirming the strict association of this psyllid species with crop availability. In 2022, 27.5% of the total tested psyllid samples resulted in being positive for Lso haplotypes D and E, the latter being prevalent. This survey revealed, for the first time in Italy, the presence of B. trigonica adults associated with Lso in carrot crops. Although this study was limited to a few fields located in one area, it provided important evidence of the risks for Lso outbreaks and prompted further research to assess the spread and incidence of the disease in apiaceous cultivations in Italy.