Publications

4358

Background: Alfalfa, Medicago sativa L. is the most important and widely grown leguminous fodder crop in temperate and tropical regions of the world. The production of alfalfa crop is limited by several biotic stresses, among which witches’ broom disease (AWB) was reported to cause significant economic losses. Methods: The phytoplasma infected alfalfa plants were collected from a central research farm, ICAR-IGFRI, Jhansi, U.P. Qualitative parameters such as crude protein content, acid detergent fibre and neutral detergent fibre were estimated in diseased and healthy plants. Phytoplasma universal primer (P1/P7) and nested primer (R16mF2/R16mR1) were used for the molecular characterization of AWB infected plants and phytoplasma infected, Parthenium hysterophorus. Result: The incidence of AWB disease ranged from 8-10%. The quantitative analysis of disease plants showed reduced plant height (-35%), fresh weight (-46.89%) and dry weight (-50.08%) compared to healthy plants. The diseased plant recorded low crude protein content (-21.38%) and higher dry matter content (+0.68%), acid detergent fibre (+33.72%) and neutral detergent fibre (+13.06%). The association of phytoplasma in diseased alfalfa and parthenium samples was confirmed by using P1/P7 and R16mF2/R16mR1 primer pair and Blastn analysis shared 99.6-100% similarity with ‘Candidatus Phytoplasma australasia’ belongs to the 16Sr group II-D.

AbstractSymbiotic microbes from the genus ‘CandidatusMegaira’ (Rickettsiales) are known to be common associates of algae and ciliates. However genomic resources for these bacteria are scarce, limiting our understanding of their diversity and biology. We therefore utilized SRA and metagenomic assemblies to explore the diversity of this genus. We successfully extracted four draft ‘Ca. Megaira’ genomes including one complete scaffold for a ‘Ca. Megaira’ and identified an additional 14 draft genomes from uncategorised environmental Metagenome-Assembled Genomes. We use this information to resolve the phylogeny for the hyper-diverse ‘Ca. Megaira’, with hosts broadly spanning ciliates, micro- and macro-algae, and find that the current single genus designation ‘Ca. Megaira’ significantly underestimates their diversity. We also evaluate the metabolic potential and diversity of ‘Ca. Megaira’ from this new genomic data and find no clear evidence of nutritional symbiosis. In contrast, we hypothesize a potential for defensive symbiosis in ‘Ca. Megaira’. Intriguingly, one symbiont genome revealed a proliferation of ORFs with ankyrin, tetratricopeptide and Leucine rich repeats like those observed in the genusWolbachiawhere they are considered important for host-symbiont protein-protein interactions. Onward research should investigate the phenotypic interactions between ‘Ca. Megaira’ and their various potential hosts, including the economically importantNemacystus decipiens, and target acquisition of genomic information to reflect the diversity of this massively variable group.Data SummaryGenomes assembled in this project have been deposited in bioproject PRJNA867165Impact statementBacteria that live inside larger organisms commonly form symbiotic relationships that impact the host’s biology in fundamental ways, such as improving defences against natural enemies or altering host reproduction. Certain groups like ciliates and algae are known to host symbiotic bacteria commonly, but our knowledge of their symbiont’s evolution and function is limited. One such bacteria is ‘CandidatusMegaira’, a Rickettsiales that was first identified in ciliates, then later in algae. To improve the available data for this common but understudied group, we searched the genomes of potential hosts on online databases for Rickettsiales and assembled their genomes. We found 4 ‘Ca. Megaira’ this way and then used these to find a further 14 genomes in environmental metagenomic data. Overall, we increased the number of known ‘Ca. Megaira’ draft genomes from 2 to 20. These new genomes show us that ‘Ca. Megaira’ is far more diverse than previously thought and that it is potentially involved in defensive symbioses. In addition, one genome shows striking resemblance to well characterized symbiont,Wolbachia, in encoding many proteins predicted to interact directly with host proteins. The genomes we have identified and examined here provide baseline resources for future work investigating the real-world interactions between the hyper diverse ‘Ca. Megaira’ and its various potential hosts, like the economically importantNemacystus decipiens.

AbstractMost prokaryotes are not available as pure cultures and therefore ineligible for naming under the rules and recommendations of the International Code of Nomenclature of Prokaryotes (ICNP). Here we summarize the development of the SeqCode, a code of nomenclature under which genome sequences serve as nomenclatural types. This code enables valid publication of names of prokaryotes based upon isolate genome, metagenome-assembled genome or single-amplified genome sequences. Otherwise, it is similar to the ICNP with regard to the formation of names and rules of priority. It operates through the SeqCode Registry (https://seqco.de/), a registration portal through which names and nomenclatural types are registered, validated and linked to metadata. We describe the two paths currently available within SeqCode to register and validate names, includingCandidatusnames, and provide examples for both. Recommendations on minimal standards for DNA sequences are provided. Thus, the SeqCode provides a reproducible and objective framework for the nomenclature of all prokaryotes regardless of cultivability and facilitates communication across microbiological disciplines.

AbstractPseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that “Candidatus Accumulibacter” can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a “Ca. Accumulibacter” enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of “Ca. Accumulibacter” contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with “Ca. Accumulibcater” and used size-exclusion chromatography to separate them into different molecular weight fractions. This separation resulted in two high molecular weight (> 5,500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in “Ca. Accumulibacter” are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This suggest that they can serve as source for sepsis treatment drugs, although further purification needs to be evaluated.Graphical abstractHighlightsNulOs in “Ca. Accumulibacter” are likely located in high molecular weight polysaccharides.Size exclusion chromatography allows to obtain high molecular weight polysaccharide-rich fractions enriched with NulOs.EPS and the NulOs-rich fractions can serve as source for sepsis treatment drugs.

Las variedades de papa (Solanum tuberosum L.) producidas en México son susceptibles a Candidatus Liberibacter solanacearum (CaLso), causante de la enfermedad conocida como ‘manchado interno de la pulpa’, por lo que se requiere conocer la respuesta de genotipos experimentales a la bacteria. El presente estudio tuvo como objetivo evaluar el efecto de la infección de los haplotipos LsoA + LsoB de CaLso en el follaje, la biomasa seca y la calidad de tubérculo de papa, variedad Fianna, una colecta de Solanum demissum y los clones experimentales T90-1-63 y T05-13-21 de Solanum spp. El manchado interno de la pulpa del tubérculo se determinó mediante análisis de imágenes de tubérculos. Las plantas de Fianna mostraron la mayor severidad de daño foliar; en cambio, los clones experimentales presentaron 17 % menos daño foliar que Fianna y 8 % más daño foliar que S. demissum. Este último fue el genotipo con la mayor biomasa seca de hoja y produjo tubérculos de un tamaño pequeño; las plantas infectadas de S. demissum presentaron mayor número de tubérculos y mayor rendimiento de tubérculo fresco que las plantas sin inoculación, aunque tuvieron la menor proporción de superficie sana del tubérculo. El clon T90-1-63 presentó los porcentajes más altos de superficie sana de tubérculo (> 79 %) y las menores intensidades del manchado interno de la pulpa del tubérculo.

Thousands of new bacterial and archaeal species and higher-level taxa are discovered each year through the analysis of genomes and metagenomes. The Genome Taxonomy Database (GTDB) provides hierarchical sequence-based descriptions and classifications for new and as-yet-unnamed taxa. However, bacterial nomenclature, as currently configured, cannot keep up with the need for new well-formed names. Instead, microbiologists have been forced to use hard-to-remember alphanumeric placeholder labels. Here, we exploit an approach to the generation of well-formed arbitrary Latinate names at a scale sufficient to name tens of thousands of unnamed taxa within GTDB. These newly created names represent an important resource for the microbiology community, facilitating communication between bioinformaticians, microbiologists and taxonomists, while populating the emerging landscape of microbial taxonomic and functional discovery with accessible and memorable linguistic labels.

Tobacco (Nicotiana tabacum) is among the agricultural products with the highest added value in Turkey. Although frequently associated with its negative effects on human health, it also provides important contributions to the Turkish economy with the employment it creates in rural areas and continues to be a strategic product. Many postgraduate theses and studies related to the sociological and economic importance of the production of this plant, which is of great importance for our country, have been carried out. However, there are very limited studies on plant diseases in tobacco production areas in Turkey. Phytoplasma is one of the important plant pathogens that cause yield loss in tobacco. Since available data on phytoplasma diseases on tobacco was very scarce worldwide, field surveys to collect samples showing phytoplasma infection-like symptoms such as yellowish color changes, leaf blisters, proliferation, dwarfism, and other physical abnormalities were carried out in Çanakkale and Balıkesir provinces of Turkey from June to August 2021. The presence of phytoplasmas in six samples was confirmed by 16S ribosomal DNA amplification by nested-PCR using universal phytoplasma primer sets, which also suggested the pathogen associated with the symptoms on tobacco. According to phylogenetic study and virtual-RFLP analysis using AluI and MseI endonuclease enzymes, the six Turkish tobacco phytoplasma strains all belong to group 16SrXII and have more than 99% nucleotide sequence identity with some members of ‘Candidatus Phytoplasma solani’ of the taxonomic subgroup ‘stolbur’ (16SrXII-A). Genetic distances analysis indicated that group 16SrI was more closely related to 16SrXII than 16SrVI, in agreement with the groups clustering in the phylogenetic tree. Neutrality tests found that 16SrI and 16SrXII groups are experiencing expanding or bottleneck selections, probably due to new mutations in the 16S rRNA gene fragment. Meanwhile, 16SrVI populations are shown to be undergoing balancing selections, indicating that its isolates have evolved for a long time.

‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’) is a crop pathogen that is a member of the 16SrXII-A ribosomal subgroup. It is also known as stolbur phytoplasma and causes yield losses in several important crops, especially in Solanaceous crops. Different strains of the pathogen are regularly reported all over the world, particularly in the Mediterranean region. In this study, the determination of genetic diversity for the pathogen infecting tomatoes and potatoes was carried out by using multilocus sequence typing analysis for the Tuf, SecY, and Vmp1 genes to gain insight into the epidemiology of ‘Ca. P. solani’ in Turkey. Genetic diversity of the phytoplasmas was investigated by sequence-based phylogenetic analyses and in silico RFLP analysis of related genes. It was determined that all ‘Ca. P. solani’-related strains infecting tomatoes and potatoes were tuf-b, which is linked to field bindweed (Convolvulus arvensis L.). Tomato or potato-infecting ‘Ca. P. solani’-related strains showed similarities with each other; however, the isolates collected from different plants showed genetic differences in terms of the SecY gene. This study indicates that the highest genetic variability of collected samples was found in the Vmp1 gene. RsaI-RFLP analysis of TYPH10F/R amplicons showed that potato-infecting ‘Ca. P. solani’-related strains were found to be similar to some existing V types. However, the V-type of tomato-infecting isolates is not similar to any previously reported V-type. The results indicate that there could be an important genetic diversity of ‘Ca. P. solani’-related phytoplasmas in Turkey. This could indicate various ways in which the pathogen has adapted to the two host plants as a consequence of the various Vmp1 gene rearrangements seen in these two plant hosts. Obtained results also indicate that the epidemiology of ‘Ca. P. solani’-related phytoplasmas in the tomato and potato agroecosystem may be better understood with the use of molecular data on the complex of vmp-types.

Huanglongbing (HLB), the most destructive citrus disease, is associated with unculturable, phloem-limited Candidatus Liberibacter species, mainly Ca. L. asiaticus (Las). Las is transmitted naturally by the insect Diaphorina citri. In a previous study, we determined that the Oceanian citrus relatives Eremocitrus glauca, Microcitrus warburgiana, Microcitrus papuana, and Microcitrus australis and three hybrids among them and Citrus were full-resistant to Las. After 2 years of evaluations, leaves of those seven genotypes remained Las-free even with their susceptible rootstock being infected. However, Las was detected in their stem bark above the scion-rootstock graft union. Aiming to gain an understanding of the full-resistance phenotype, new experiments were carried out with the challenge-inoculated Oceanian citrus genotypes through which we evaluated: (1) Las acquisition by D. citri fed onto them; (2) Las infection in sweet orange plants grafted with bark or budwood from them; (3) Las infection in sweet orange plants top-grafted onto them; (4) Las infection in new shoots from rooted plants of them; and (5) Las infection in new shoots of them after drastic back-pruning. Overall, results showed that insects that fed on plants from the Oceanian citrus genotypes, their canopies, new flushes, and leaves from rooted cuttings evaluated remained quantitative real-time polymerase chain reaction (qPCR)-negative. Moreover, their budwood pieces were unable to infect sweet orange through grafting. Furthermore, sweet orange control leaves resulted infected when insects fed onto them and graft-receptor susceptible plants. Genomic and morphological analysis of the Oceanian genotypes corroborated that E. glauca and M. warburgiana are pure species while our M. australis accession is an M. australis × M. inodora hybrid and M. papuana is probably a M. papuana × M. warburgiana hybrid. E. glauca × C. sinensis hybrid was found coming from a cross between E. glauca and mandarin or tangor. Eremocitrus × Microcitrus hybrid is a complex admixture of M. australasica, M. australis, and E. glauca while the last hybrid is an M. australasica × M. australis admixture. Confirmation of consistent full resistance in these genotypes with proper validation of their genomic parentages is essential to map properly genomic regions for breeding programs aimed to generate new Citrus-like cultivars yielding immunity to HLB.

Abstract The candidate phyla radiation (CPR) has been described as an obligatory group of ultrasmall bacteria associated with host bacteria. They phylogenetically represent a subdivision of bacteria distinct from other living organisms. Using polyphasic approaches, we screened human faecal samples for the detection of Saccharibacteria. The new sequences obtained by sequencing were compared to the complete CPR genomes available to date. Then, we attempted a co-culture of CPR-bacteria and non-CPR bacteria from human faecal samples. We finally aimed to evaluate the prevalence and distribution of these Saccharibacteria sequences in human sources in 16S amplicon datasets. We were able to reconstitute two high-quality Saccharibacteria genomes named Minimicrobia massiliensis and Minimicrobia timonensis. We have established, for the first time in human digestive samples, the coculture of Candidatus Saccharibacteria with two different bacterial hosts. Finally, we showed that 12.8% (610/4,756) of samples sequenced in our laboratory were positive for operational taxonomic units (OTUs) assigned to M.massiliensis. and significantly enriched in human respiratory and oral microbiota. Here, we reported the first genomes and coculture of Saccharibacteria from human gut specimens. This study opens a new field, particularly in the study of the involvement of CPR in the human intestinal microbiota.