Chryseobacterium paridis sp. nov., an endophytic bacterial species isolated from the root of Paris polyphylla Smith var. yunnanensis

A Gram-negative, yellow-pigmented, rod-shaped bacterial strain YIM B02567T was isolated from the root of Paris polyphylla Smith var. yunnanensis in China. Strain YIM B02567T grew optimally at 25–30 °C and at pH 7.0 in the absence of NaCl on nutrient agar. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain YIM B02567T belong to the genus Chryseobacterium, and was closely related to Chryseobacterium piperi CTMT and Chryseobacterium soli DSM 19298T. Whole genome sequencing indicated that the genome size was 4,774,612 bp and with a G + C content of 34.5 mol%. Values of the ANI and the dDDH between strain YIM B02567T and its closely related Chryseobacterium species were below 81.72% and 24.7%. Strain YIM B02567T contained menaquinone-6 as the sole isoprenoid quinone, anteiso-C15:0, iso-C17:1ω9c and iso-C17:0 3-OH as major fatty acids and phosphatidylethanolamine as major polar lipid. Based on the polyphasic analyses, strain YIM B02567T could be differentiated genotypically and phenotypically from recognized species of the genus Chryseobacterium. The isolate, therefore, represents a novel species, for which the name Chryseobacterium paridis sp. nov. is proposed. The type strain is YIM B02567T (= CGMCC 1.18657T).


Introduction
The genus Chryseobacterium is a member of the family Flavobacteriaceae in the phylum Bacteroidetes. It was proposed by Vandamme et al. (1994) to separate six species of the genus Flavobacterium, based on the genotypic, biochemical and phenotypic characteristics of the organisms. At the time of writing, more than 100 valid published species of the genus Chryseobacterium have been reported (https:// lpsn. dsmz. de/ genus/ chrys eobac terium); many of these are abundant in diverse environments, including soil (Benmalek et al. 2010), water (Montero-Calasanz et al. 2013), plants (Du et al. 2015), rhizospheres (Park et al. 2006), raw milk (Hantsis-Zacharov and Halpern 2007), chicken (Kämpfer et al. 2014) and fish (Ilardi et al. 2009). Interestingly, several species of the genus Chryseobacterium, were isolated from plants or rhizospheres, hence this genus contains several rhizospheric species. Such as, Chryseobacterium cucumeris isolated from cucumber (Cucumis sativus L.) root (Jeong et al. 2017), Chryseobacterium ginsengisoli isolated from the rhizosphere of ginseng (Nguyen et al. 2013), and Chryseobacterium ginsenosidimutans isolated from soil of a Rhus vernicifera-cultivated field (Im et al. 2011). In this 1 3 study, we described a new species of the genus Chryseobacterium, designated YIM B02567 T , isolated from the root of Paris polyphylla var. yunnanensis.

Bacterial isolation and maintenance
Healthy root samples of P. polyphylla var. yunnanensis were collected from Shilin in Yunnan province, southwest PR China. Samples were sterilized and pulverized before distribution on nutrient agar (NA) medium as described by Yang et al. (2016). After incubation at 28 °C for 2 weeks, different colonies were randomly selected and their 16S rRNA genes were PCR-amplified. Strain YIM B02567 T was selected as a putative novel species of the genus Chryseobacterium for further taxonomic characterizations. The purified strain was preserved both on NA slants at 4 °C and in 20% (v/v) glycerol at − 80 °C for further use.

16S rRNA gene sequencing and phylogenetic analysis
Genomic DNA of strain YIM B02567 T was extracted using a genomic DNA extraction kit (Tiangen, China). The 16S rRNA gene was amplified by PCR using forward primer 27F (5′-AGA GTT TGA TCC TGG CT-3′) and reverse primer 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). Amplified products were purified and cloned into vector pClone007 (TsingKe, China). The 16S rRNA gene sequence (1536 bp) of strain YIM B02567 T was checked manually and submitted to the GenBank database. The similarities of 16S rRNA gene sequences between strain YIM B02567 T and closely related type strains were calculated using the EZBioCloud server (https:// www. ezbio cloud. net/) (Yoon et al. 2017). The 16S rRNA gene sequences were aligned by using Clustal Omega (Sievers et al. 2011) software and Kimura's twoparameter model (Kimura 1980). Phylogenetic trees were constructed with the Neighbor-joining (NJ) (Saitou and Nei 1987), Maximum-likelihood (ML) (Felsenstein 1981) methods using Mega X software (Kumar et al. 2018). Bootstrap analysis with 1000 replicates was conducted to assess confidence levels for the branches (Felsenstein 1985).

Whole genome sequencing and analysis
The whole-genome sequencing of strain YIM B02567 T was performed using BGISEQ platform by China General Microbiological Culture Collection Center (CGMCC) as part of the Global Catalogue of Microorganisms (GCM) 10 K project (Shi et al. 2021). The sequence data were assembled using SOAPdenovo 2.04 (Luo et al. 2012). The average nucleotide identity (ANI) values between strains YIM B02567 T and reference strains were calculated using FastANI (Jain et al. 2018). Average amino acid identity (AAI) values were calculated from protein sequences using an online AAI calculator (http:// enve-omics. ce. gatech. edu/ aai/). The estimated genome-sequence-based digital DNA-DNA hybridization (dDDH) values were calculated using formula 2 at the Genome-to-Genome Calculator (CGGC) website (https:// ggdc. dsmz. de/ ggdc. php) as described by Meier-Kolthoff et al. (2013).
For further confirming the taxonomy status, phylogenomic analysis of strain YIM B02567 T and related species was performed. Genome sequences of the related species' type strains were collected from NCBI GenBank Database. And all of these genomes were annotated using PROKKA (Seemann 2014). The orthologous gene inferring was using by OrthoFinder (Emms and Kelly 2015). The selected orthologs were aligned using the Clustal Omega (Sievers et al. 2011) and concatenating all alignments. Gblocks (Castresana 2000) was used to select the conserved blocks from the concatenation. The reconstruction of a ML tree was using IQ tree (Nguyen et al. 2015).

Morphology and physiology and biochemical analysis
Cell morphology was observed by scanning electron microscope after growth for 2 days in Reasoner's 2A (R2A) medium at 30 °C. The Gram reaction was performed using 3% (w/v) KOH for cell lysis. The growth of the strain was assessed by incubating inoculated R2A plates in a bacteria culture box at 30 °C for 7 days. Growth was examined at different temperatures (low to 20 °C, up to 50 °C, at intervals of 5 °C) and NaCl concentrations (up to 5.0%, at intervals of 0.5%, w/v) for 7 days. The pH range for growth was tested between 4.0 and 10.0, at intervals of 1.0 pH unit in R2A broths prepared using the buffer system described by Nie et al. (2012). Catalase activity was determined from the production of gas bubbles on the addition of a drop of 3% (v/v) H 2 O 2 . Oxidase activity was detected using API oxidase reagent (bioMérieux) according to the manufacturer's instructions. Carbon source utilization was checked in Biolog GENIII microplates. Additional biochemical characteristics and enzymatic activities were further determined using the API 20NE and API ZYM kits (bioMérieux) according to the instructions provided by the manufacturers.

Chemotaxonomic characterization
The fatty acid profile, polar lipids and respiratory quinones of strain YIM B02567 T were analyzed in this study. To assess the fatty acids, strain YIM B02567 T were cultured on R2A agar plates at 30 °C for 2 days. After saponification and methylation, fatty acids were extracted using a standard protocol and the Sherlock Microbial Identification (Sherlock version 6.1; MIDI database: TSBA6) according to the manufacturer's instructions (Sasser 2001) and analyzed on Agilent 7890A gas chromatography apparatus. Respiratory quinones and polar lipids were extracted from freeze-dried cells using the method described by Collins et al. (1977). Subsequently, quinones were analyzed by a reversed-phase HPLC system (Agilent Technologies 1260 Infinity) with a C18 column (25 cm × 4.6 mm, 5 μm). Extracted total lipids from strain YIM B02567 T were examined by a two-dimensional TLC procedure on silica gel G60 plates (Minnikin et al. 1984).
For the presence of all lipids, TLC plates were sprayed with 5% molybdophosphoric acid. Besides, 0.2% ninhydrin was used to detect aminolipids, molybdenum blue spray reagent was used to detect phospholipids and a-naphthol reagent was used to detect glycolipids.

Phylogenetic and whole-genome analysis
The 16S rRNA gene sequence analysis based on EzTaxon server revealed that strain YIM B02567 T belonged to the genus Chryseobacterium and had highest gene sequence similarities to C. soli DSM 19298 T (97.8%), C. ginsenosidimutans THG 15 T (97.7%), Chryseobacterium soldanellicola DSM 17072 T (97.5%) and C. piperi CTM T (97.4%). The NJ phylogenetic tree based on 16S rRNA gene sequences showed that strain YIM B02567 T , C. soli and C. piperi formed a monophyletic clade (Fig. 1), but did not clustered with C. ginsenosidimutans and C. soldanellicola. This topology relationship was supported by the ML tree. (Fig. S3). Furthermore, a ML phylogenomic tree reconstructed using 1164 orthologous genes confirm that strain YIM B02567 T is most closely with C. piperi CTM T (Fig. 2).
The draft genome of strain YIM B02567 T contained 12 scaffolds, with a total length of 4,774,612 bp and the N50 length of 2,588,358 bp. The DNA G + C content of strain YIM B02567 T was determined from the genome to be 34.5 mol%. The annotated result of YIM B02567 T  Table S1. Strain YIM B02567 T has AAI values ranging from 78.77% to 86.67% with the all reference genomes (Table S1). The ANI and AAI values were significantly lower than the widely accepted threshold for describing prokaryote species (95-96%; Kim et al. 2014;Konstantinidis and Tiedje 2005). The dDDH values of strain YIM B02567 T to C. piperi CTM T and C. soli DSM 19298 T were 24.7% and 22.2%, which were significantly lower than 70% similarity of the species defined threshold (Wayne et al. 1987). Therefore, according to the results of OGRIs (overall genome relatedness indices), strain YIM B02567 T can represent a novel species of the genus Chryseobacterium.

Morphology, physiology and biochemical analysis
Cells of strain YIM B02567 T were Gram-reaction-negative, aerobic, oxidase-and catalase-positive and rodshaped (Fig. S1). Colonies on R2A agar were deep orange and smooth after incubation at 30 °C for 2 days. Strains were able to grow at temperatures ranging between 10 and 45 °C, pH 5.0-8.0 and in the presence of up to 2.0% (w/v) NaCl on R2A. Detail physiological and biochemical characteristics of the novel strain were summarized in the species description and compared to those of closely related strains in Table 1.

Chemotaxonomic characterization
The major cellular fatty acids of strain YIM B02567 T were iso-C 15:0 (41.8%), iso-C 17:0 3-OH (16.9%), iso-C 17:1 ω9c (14.0%), Summed Feature 3 (C 16:1 ω5c and/or C 16:1 ω6c, 13.9%). Strain YIM B02567 T showed a similar major fatty acid composition to the related type strains of Chryseobacterium species. However, some qualitative and quantitative differences in the fatty acid compositions were observed between the novel strain and the other closely related Chryseobacterium species (Table 2). The major polar lipids of strain YIM B02567 T was phosphatidylethanolamine (PE).Three unidentified aminolipids (AL), five unidentified glycolipids (GL) and three unidentified lipids (L) were also detected (Fig. S2). The predominant sole respiratory ubiquinone was found to be MK-6, which is the typical ubiquinone of the genus Chryseobacterium.

Taxonomic conclusion
Based on morphological, physiological, and chemotaxonomic properties, and phylogenetic analysis, strain YIM B02567 T could be considered a representative of a novel species belonging in the genus Chryseobacterium, for which the name Chryseobacterium paridis sp. nov. is proposed.

Description of Chryseobacterium paridis sp. nov.
Chryseobacterium paridis (pa'ri.dis. L. gen. n. paridis of Paris, a plant genus, from which the type strain was isolated).