RESUMO
The Gram-positive genus Clavibacter is currently divided into seven species (Clavibacter michiganensis, Clavibacter nebraskensis, Clavibacter capsici, Clavibacter sepedonicus, Clavibacter tessellarius, Clavibacter insidiosus and Clavibacter zhangzhiyongii) and three subspecies (C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli). Recent studies have indicated that the taxonomic rank of the subspecies must be re-evaluated. In this research, we assessed the taxonomic position of the three C. michiganensis subspecies and clarified the taxonomic nomenclature of other 75 Clavibacter strains. The complete genomes of the type strains of the three Clavibacter subspecies, the type strain of C. tessellarius and C. nebraskensis A6096 were sequenced using PacBio RSII technology. Application of whole-genome-based computational approaches such as average nucleotide identity (ANI), digital DNA-DNA hybridization, multi-locus sequence analysis of seven housekeeping genes (acnA, atpD, bipA, icdA, mtlD, recA and rpoB), a phylogenomic tree reconstructed from 1â028 core genes, and ANI-based phylogeny provided sufficient justification for raising C. michiganensis subsp. californiensis to the species level. These results led us to propose the establishment of Clavibacter californiensis sp. nov. as a species with its type strain C55T (=CFBP 8216T=ATCC BAA-2691T). Moreover, the orthologous and in silico dot plot analyses, along with the above described bioinformatic strategies, revealed a high degree of similarity between C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli. Based on these analyses, we propose that both subspecies be combined into a single taxon and elevated to the species level as Clavibacter phaseoli sp. nov., with LPPA 982T (=âCECT 8144T=âLMG 27667T) as the type strain.
Assuntos
Clavibacter , Ácidos Graxos , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Nucleotídeos , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
A autora apresenta duas condições bem diferentes que interferem na capacidade de uma pessoa vivenciar o luto: se lamuriar muito e se lamuriar muito pouco. Desde 1917, cinco estados mentais específicos, presentes na descrição da melancolia feita por Freud, foram aprofundados por teóricos subsequentes. Neste artigo a autora concentra-se no quarto e no quinto estado. Menciona os objetos não valorizados em oposição aos objetos valorizados. Considera que todos os desenvolvimentos mais recentes do pensamento psicanalítico se apoiam na grande atenção à teoria das relações objetais e na crescente compreensão da natureza da diferença entre os processos patológicos e benignos projetivos e os processos patológicos e benignos introjetivos.
The author presents two very different conditions, which interfere with a person's capacity to mourn: whimpering too much and whimpering too little. Since 1917, five particular states of mind, which appear in Freud'd description of melancholia, have been developed further by subsequent theorists. This study focuses on the fourth and fifth states. It mentions the unvalued objects, as opposed to the valued objects. It also states that all the newer developments in psychoanalytic thinking are underpinned by the greater attention to the object-relations theory, and by the growing understanding of the nature of the difference between the pathological and benign projective processes, and pathological and benign introjective ones.
La autora presenta dos condiciones muy distintas que interfieren en la capacidad que una persona tiene de vivir el luto: lloriquear mucho y lloriquear muy poco. Desde 1917, cinco estados mentales específicos, presentes en la descripción de la melancolía hecha por Freud, fueron examinados más a fondo por teóricos posteriores. En este artículo la autora se centra en el cuarto y en el quinto estado. Menciona los objetos no apreciados en oposición a los objetos apreciados. Considera que todos los desarrollos más recientes del pensamiento psicoanalítico se respaldan en la gran atención a la teoría de las relaciones objetales en la creciente comprensión de la naturaleza de la diferencia entre os procesos patológicos y benignos proyectivos y los procesos patológicos y benignos introyectados.
L'autrice présente deux conditions très différentes qui interfèrent avec la capacité d'une personne vivre le deuil : se plaindre trop et se plaindre très peu. Depuis 1917, cinq stages mentaux spécifiques, compris dans la description faite par Freud de la mélancolie ont été approfondis par les théoriques qui viennent après lui. Dans cet article l'autrice se focalise sur le quatrième et cinquième stage. Elle mentionne les objets non valorisés en opposition à ceux valorisés. Elle estime que tous les développements les plus récents de la pensée psychanalytique se fondent sur la grande attention donnée à la théorie des relations objectales et sur la compréhension croissante de la nature de la différence entre les processus pathologiques et les processus pathologiques et bénins projectifs et les processus pathologiques et bénins d'introjection.
RESUMO
Mizuna (Brassica rapa var. japonica), a member of family Brassicaceae, is a leafy vegetable having phenolic and other compounds beneficial to human health, such as natural antioxidants (Khanam et al. 2012). In October 2020, a field of mizuna (variety: Early) on Oahu island was observed having 20-30% diseased plants. Four randomly selected infected mizuna plants, showing the symptoms of wilt and stem rot (Figure 1A-D), were collected and isolations were made to determine the pathogen. Small sections of infected stems were cut, surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The tissues were macerated in a sterile 1.5 ml centrifuge tube containing 100 µl sterile water-macerated tissues were streaked onto crystal violet pectate medium (CVP) (Hélias et al. 2011) and incubated at 26 ± 2°C for 48 h. Isolated bacterial colonies that formed pits on the CVP plates were re-streaked onto dextrose peptone agar: Peptone (10 g/L), Dextrose (5 g/L) and Agar (17 g/L) (DPA-without tetrazolium chloride; Norman and Alvarez 1989) to obtain purified colonies for DNA isolation using DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA). The two housekeeping genes (dnaA and gapA) were amplified and sequenced following the protocols used by Dobhal et al. (2020) and Boluk et al. (2020), for identity confirmation and phylogenetic analysis. Cleaned PCR products were sent to the GENEWIZ facility (Genewiz, La Jolla, CA) for sequencing of sense and antisense strands. The obtained sequences were aligned, manually edited, and consensus sequences were analyzed with BLASTn using the NCBI GenBank nucleotide and genome databases for identity confirmation. The BLASTn results demonstrated 100% query coverage of all four strains (PL248-PL251); and showed 100% identity of PL248 and PL249, and 99% identity of PL250 and PL251 with Pectobacterium brasiliense. All the sequences were submitted to the NCBI GenBank database under the following accession numbers: dnaA gene MW560271 - MW560274 (PL248 - PL251); and gapA gene MW560275 - MW560278 (PL248 - PL251). Pathogenicity was assessed by artificially inoculating 100 µl bacterial suspension of each strain (PL248 - 1.12x 108 CFU/ml; PL249 - 1.32x 108 CFU/ml; PL 250 - 1.2x 108 CFU/ml and PL251 - 1.15x 108 CFU/ml) onto four-week-old mizuna (variety: Leafy Asian Greens) plants in three replicates, using sterile pipette tips, which was stabbed into stem halfway and wrapped with parafilm. The inoculated plants were well maintained under controlled greenhouse conditions. As negative controls, three plants were inoculated with 100 µl distilled water. Soft rot and wilt symptoms (Figure 1E-H) were observed 24 hours post inoculation. No symptoms were observed on control plants (Figure 1F). All four strains were re-isolated from the inoculated plants and confirmed as P. brasiliense based on resequencing of the dnaA region and 100% homology with the sequences of original strain. In the phylogenetic tree (Figure 2), based on two housekeeping genes (dnaA and gapA), the bacterial strains from mizuna grouped with other P. brasiliense retrieved from the NCBI GenBank database. To our knowledge, this is the first report of P. brasiliense infecting mizuna plants in Hawaii or in the USA and is important because this species is one of the most aggressive pectolytic pathogens in the genus Pectobacterium. Understanding the diversity of different pectolytic phytopathogens is essential to formulating risk mitigation strategies as P. brasiliense could potentially pose a threat to additional vegetable crops, especially the crucifers vegetables (Arizala et al. 2019; Klair et al, 2021).