Publications

4373

Abstract‘Candidatus Phytoplasma ulmi’, associated with elm yellows, shoot proliferation and dieback of elm (Ulmus) species trees was reported in United States of America and in many European countries. Until now its presence in elm trees in Poland has not been detected. In 2017–2018, during visual inspection of elm trees grown in four areas of southern Silesia Province leaf yellowing, shoot proliferation, phloem necrosis and dieback of branches were observed on European field elm trees grown in nature reserve close to the border with the Czech Republic. Phloem tissue from shoots collected from six symptomatic and twenty asymptomatic trees was used for extraction nucleic acids. Nested PCR with primer pairs amplifying 16Sr RNA sequences of phytoplasmas resulted in specific products for samples from six symptomatic trees however the restriction fragment length polymorphism (RFLP) and phylogenetic analyses of the sequenced products did not allow to clearly classify newly detected isolates within the elm yellows group (16SrV). The isolates found in Poland clustered on the boundary of the 16SrV‐A and 16SrV‐C ribosomal subgroups. Sequence analyses of less conservative genes rpl22‐ rps3 and secY confirmed the affiliation of the phytoplasma isolates found in Poland to ‘Candidatus Phytoplasma ulmi’; however, the significant nucleotide changes in signature sequences for all three genes were shown compared with EY1T reference strain. Surprisingly, the newly detected isolates shared a common origin and were phylogenetically closer to EY1‐SRB and EY10‐SRB strains from Serbia as well as NK16 strain from Croatia strains than to the EYCZ1 strain from the Czech Republic. This is the first report on detection and molecular characterization of ‘Candidatus Phytoplasma ulmi’ in Poland based on sequence analysis of three genetic locus.

Summary Complete genomes can be recovered from metagenomes by assembling and binning DNA sequences into metagenome assembled genomes (MAGs). Yet, the presence of microdiversity can hamper the assembly and binning processes, possibly yielding chimeric, highly fragmented and incomplete genomes. Here, the metagenomes of four samples of aerobic granular sludge bioreactors containing Candidatus ( Ca .) Accumulibacter, a phosphate‐accumulating organism of interest for wastewater treatment, were sequenced with both PacBio and Illumina. Different strategies of genome assembly and binning were investigated, including published protocols and a binning procedure adapted to the binning of long contigs (MuLoBiSC). Multiple criteria were considered to select the best strategy for Ca . Accumulibacter, whose multiple strains in every sample represent a challenging microdiversity. In this case, the best strategy relies on long‐read only assembly and a custom binning procedure including MuLoBiSC in metaWRAP. Several high‐quality Ca . Accumulibacter MAGs, including a novel species, were obtained independently from different samples. Comparative genomic analysis showed that MAGs retrieved in different samples harbour genomic rearrangements in addition to accumulation of point mutations. The microdiversity of Ca . Accumulibacter, likely driven by mobile genetic elements, causes major difficulties in recovering MAGs, but it is also a hallmark of the panmictic lifestyle of these bacteria.

The Synechococcales is a large cyanobacterial order comprising both unicellular and filamentous forms, with parietal thylakoid arrangement. Previously, this order has been the subject of taxonomic revisions with new families being erected. During studies of the phototrophic communities on the limestone walls of the Old Cathedral of Coimbra (UNESCO monument), a coccoid Aphanocapsa‐like cyanobacterium was isolated. It was characterized using a polyphasic approach, based on morphology, 16S rRNA phylogenetic and phylogenomic analyses, internal transcribed spacer (ITS) secondary structure, and ecology. The 16S rRNA phylogenetic analyses showed that this strain is placed in a separate and highly supported family‐level clade, as part of a large group comprising the families Prochlorococcaceae and Prochlorotrichaceae, with Lagosinema as the closest (although quite distant) taxon. Additionally, the phylogenomic analysis also placed this strain in a separate lineage, situated distantly apart from the family Thermosynechococcaceae, but with strains assigned to Acaryochloris marina MBIC 11017 and Aphanocapsa montana BDHKU210001 as the closest taxa. Based on these data, as well as on the results from the secondary ITS structure, morphology, and ecology, we here propose the establishment of Petrachlorosaceae fam. nov., along with the description of Petrachloros gen. nov. and Petrachloros mirabilis sp. nov. We also address additional considerations regarding some cyanobacterial taxa within the order Synechococcales, which we believe deserve further revisions.

AbstractAn actinobacterial strain A23T, isolated from adult ant Camponotus vagus collected in Ryazan region (Russia) and established as tetracenomycin X producer, was subjected to a polyphasic taxonomic study. Morphological characteristics of this strain included well-branched substrate mycelium and aerial hyphae fragmented into rod-shaped elements. Phylogenetic analyses based on 16S rRNA gene and genome sequences showed that strain A23T was most closely related to Amycolatopsis pretoriensis DSM 44654T. Average nucleotide identity and digital DNA–DNA hybridization values between the genome sequences of isolate A23T and its closest relative, Amycolatopsis pretoriensis DSM 44654T, were 39.5% and 88.6%, which were below the 70% and 95–96% cut-off point recommended for bacterial species demarcation, respectively. The genome size of the isolate A23T was 10,560,374 bp with a DNA G + C content of 71.2%. The whole-cell hydrolysate contained meso-diaminopimelic acid and arabinose and galactose as main diagnostic sugars as well as ribose and rhamnose. It contained MK-9(H4) as the predominant menaquinone and iso-C16:0, iso-C15:0, anteiso-C17:0 and C16:0 as the major cellular fatty acids. Diphosphatidylglycerol and phosphatidylethanolamine prevailed among phospholipids. Mycolic acids were not detected. Based on the phenotypic, genomic and phylogenetic data, isolate A23T represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis camponoti sp. nov. is proposed, and the type strain is A23T (= DSM 111725T = VKM 2882T).

AbstractPeach trees showing witches’‐broom disease symptoms in the northwest of Iran were sampled for phytoplasma detection. PCR assays and Sanger sequence analyses indicated that ʻCandidatus Phytoplasma phoeniciumʼ was associated with peach witchesʼ‐broom disease. Virtual RFLP analyses of the 16S rRNA gene indicated that ʻCa. Phytoplasma phoeniciumʼ strain, which was prevalent in the northwest of Iran belonged to 16SrIX‐C subgroup. For the genomic characterization of Iranian ʻCa. Phytoplasma phoeniciumʼ strain, total DNA extracted from the infected peach trees was subjected to Illumina next‐generation sequencing. The NGS sequencing resulted in 41157647 read pairs of raw data. The raw reads length was 150 bp and the insert size was 350 bp. Assembly and genes clustering finally resulted in 750 contigs with a total size of 228345 bp. The GC content of sequence was 33.2%, N50 value was 259 and L50 value was 256. According to NCBI‐ PGAP annotation the genome of Prunus persica phytoplasma PP2 contained 410 genes including 377 CDSs with protein, 10 rRNAs, nine tRNAs and 14 pseudogenes.

Changes in physiological and biochemical patterns in lucerne plants caused by the presence of ‘Candidatus Phytoplasma australasia’, which is one of the significant pathogens causing yield losses in lucerne plants, were investigated. Significant differences were evident in total chlorophyll, chlorophyll a, chlorophyll b, and protein amounts between ‘Ca. Phytoplasma australasia’-positive and negative lucerne plants. Stress-related metabolites such as phenol, malondialdehyde, and proline accumulations in ‘Ca. Phytoplasma australasia’-positive plants were remarkably higher than those of phytoplasma-negative plants. As a response to disease attack, phytoplasma-positive plants exhibited higher antioxidant enzymes (peroxidase and catalase) and non-enzymatic metabolite responses such as jasmonic and salicylic acids. We state that partial disease responses were revealed for the first time to breed resistant lucerne lines infected by ‘Ca. Phytoplasma australasia’.